Method of assessing the effectiveness of a non-nucleoside reverse transcriptase inhibitor (NNRTI) on a human immunodeficiency virus type 1 (HIV-1)-infected patient

ABSTRACT

This invention relates to methods for assessing the effectiveness of non-nucleoside reverse transcriptase inhibitor on an HIV that rely on detection of combinations of mutations in HIV reverse transcriptase associated with altered susceptibility to such compounds. In some embodiments, the combinations of mutations include various combinations of mutations at codons 190, 101, 130, and 98 of HIV reverse transcriptase.

This is a divisional of application Ser. No. 09/320,299 filed May 26,1999, now U.S. Pat. No. 7,037,644 which claims the benefit of U.S.Provisional Application No. 60/086,834, filed May 26, 1998, and U.S.Provisional Application No. 60/124,090, filed Mar. 12, 1999, thecontents of which are each hereby incorporated by reference into thisapplication.

Throughout this application, various references are referred to withinparenthesis. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

TECHNICAL FIELD

This invention relates to antiretroviral drug susceptibility andresistance tests to be used in identifying effective drug regimens forthe treatment of human immunodeficiency virus (HIV) infection andacquired immunodeficiency syndrome (AIDS). The invention further relatesto the means and methods of monitoring the clinical progression of HIVinfection and its response to antiretroviral therapy using phenotypic orgenotypic susceptibility assays. The invention also relates to novelvectors, host cells and compositions for carrying out phenotypicsusceptibility tests. The invention further relates to the use ofvarious genotypic methodologies to identify patients whose infection hasbecome resistant to a particular antiretroviral drug regimen. Thisinvention also relates to the screening of candidate antiretroviraldrugs for their capacity to inhibit viruses, selected viral sequencesand/or viral proteins. More particularly, this invention relates to thedetermination of non-nucleoside reverse transcriptase inhibitorresistance using phenotypic susceptibility tests and/or genotypic tests.

BACKGROUND OF THE INVENTION

HIV infection is characterized by high rates of viral turnoverthroughout the disease process, eventually leading to CD4 depletion anddisease progression. Wei X, Ghosh S K, Taylor M E, et al. (1995) Nature343, 117-122 and Ho D D, Naumann A U, Perelson A S, et al. (1995) Nature373, 123-126. The aim of antiretroviral therapy is to achievesubstantial and prolonged suppression of viral replication. Achievingsustained viral control is likely to involve the use of sequentialtherapies, generally each therapy comprising combinations of three ormore antiretroviral drugs. Choice of initial and subsequent therapyshould, therefore, be made on a rational basis, with knowledge ofresistance and cross-resistance patterns being vital to guiding thosedecisions. The primary rationale of combination therapy relates tosynergistic or additive activity to achieve greater inhibition of viralreplication. The tolerability of drug regimens will remain critical,however, as therapy will need to be maintained over many years.

In an untreated patient, some 10¹⁰ new viral particles are produced perday. Coupled with the failure of HIV reverse transcriptase (RT) tocorrect transcription errors by exonucleolytic proofreading, this highlevel of viral turnover results in 10⁴ to 10¹⁰ mutations per day at eachposition in the HIV genome. The result is the rapid establishment ofextensive genotypic variation. While some template positions or basepair substitutions may be more error prone (Mansky L M, Temin H M (1995)J Virol 69, 5087-5094) (Schinazi R F, Lloyd R M, Ramanathan C S, et al.(1994) Antimicrob Agents Chemother 38, 268-274), mathematical modelingsuggests that, at every possible single point, mutation may occur up to10,000 times per day in infected individuals.

For antiretroviral drug resistance to occur, the target enzyme must bemodified while preserving its function in the presence of the inhibitor.Point mutations leading to an amino acid substitution may result inchange in shape, size or charge of the active site, substrate bindingsite or surrounding regions of the enzyme. Mutants resistant toantiretroviral agents have been detected at low levels before theinitiation of therapy. (Mohri H, Singh M K, Ching W T W, et al. (1993)Proc Natl Acad Sci USA 90, 25-29) (Nájera I, Richman D D, Olivares I, etal. (1994) AIDS Res Hum Retroviruses 10, 1479-1488) (Nájera I, HolguinA, Quiñones-Mateu E, et al. (1995) J Virol 69, 23-31). However, thesemutant strains represent only a small proportion of the total viral loadand may have a replication or competitive disadvantage compared withwild-type virus. (Coffin J M (1995) Science 267, 483-489). The selectivepressure of antiretroviral therapy provides these drug-resistant mutantswith a competitive advantage and thus they come to represent thedominant quasispecies (Frost S D W, McLean A R (1994) AIDS 8, 323-332)(Kellam P, Boucher C A B, Tijnagal J M G H (1994) J Gen Virol 75,341-351) ultimately leading to drug resistance and virologic failure inthe patient.

Non-Nucleoside Reverse Transcriptase Inhibitors

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are achemically diverse group of compounds which are potent inhibitors ofHIV-1 RT in vitro. These compounds include pyridinone derivatives,bis(heteroaryl)piperazines (BHAPs) such as delavirdine and atevirdine,the dipyridodiazepinone nevirapine, the thymine derivative groups TSAOand HEPT, an α-anilino phenylacetamides (α-APA) compound loviride, andthe quinoxaline-class inhibitors such as (HBY-097), thebenzodiazepin-one and thione (TIBO) compounds and the pyridinonederivatives (L-697,661). For overviews see (DeClercq E. (1996) Rev MedVirol 6, 97-117) (Emini E A (1996) Antiviral Drug Resistance, ed. D DRichman, John Wiley & Sons, Ltd. High-level resistance to individualcompounds appears to develop rapidly, often within a few weeks ofinitiating monotherapy, frequently involving only single-point mutationsand in many cases leading to considerable cross-resistance to otherNNRTIs. Most mutations reported occur in the codon groups 100-108 and181-190 which encode for the two β-sheets adjacent to the catalytic siteof the RT enzyme (Kohlstaedt L A, Wang J, Friedman J M, et al. (1992)Science 256, 1783-90). The NNRTI binding pocket, as it has beendescribed, is a hydrophobic non-substrate binding region of RT wherethese agents directly interact with RT. They inhibit activity byinterfering with mobility of the ‘thumb’ subdomain, or disrupting theorientation of conserved aspartic acid side chains essential forcatalytic activity (D'Aquilla R T. (1994) Clin Lab Med 14, 393-423)(Arnold E., Ding J., Hughes S H, et al. (1995) Curr Opin Struct Biol 5,27-38).

Mutations conferring reduced susceptibility to nevirapine have beendescribed at codons 98, 100, 103, 106, 108, 181, 188 and 190 (Richman DD, Havlir D, Corbeil J. (1994) J Virol 68, 1660-1666). The mostfrequently selected variant during nevirapine monotherapy is aTyr181_Cys (Y181C) mutation which results in a 100-fold reduction insensitivity to this agent, with reduced susceptibility to the pyridinonederivatives L-696,229 and L-697,661 (Arnold, Ibid). TSAO also haslimited activity in the presence of the 181 mutation, but maintainsactivity in the presence of mutations at codons 100 and 103 and in vitroselects for a unique mutation, GLU138_Lys (E138K), in the region whereit most closely interacts with RT (Richman D D, Ibid) (Richman D D, ShihC-K, Lowy I, et al. (1991) Proc Natl Acad Sci USA 88, 11241-11245).

Resistance to loviride when used as monotherapy develops in mostpatients by week 24. It has been mapped to a range of codons 100-110;181-190), most commonly codon 103 (Staszewski S, Miller V, Kober A, etal. (1996) Antiviral Ther 1, 42-50). During combination therapy usingloviride with zidovudine or zidovudine plus lamivudine, variants atcodons 98 and 103 were the most frequent mutations detected at 24 weeks(Staszewski S, Miller V, Rehmet S, et al. (1996) AIDS 10, F1-7).

Although the 101, 103 and 181 mutations also confer cross-resistance toBHAPs. (Balzarini J, Karlsson A, Pérez-Pérez M-J, et al. (1992) Virology192, 246-253) the characteristic P236L substitution selected for bythese agents in vitro appears to sensitize RT to some other NNRTIs,reducing the IC50 for nevirapine, for example, 7- to 10-fold, withoutinfluencing sensitivity to nucleoside analogues (Staszewski S, Ibid).This mutation at codon 236 has not been observed in clinical isolatesduring atevirdine therapy, although other resistance-conferringmutations at codons 103 and 181 have been reported during monotherapy aswell as at codons 101, 188, 233 and 238 during combination therapy withzidovudine.

While HBY-097 may initially select for a mutation at codons 190 invitro, further passage consistently selects for mutations at RT codon 74and 75, with some mutant viruses showing decreased sensitivity todidanosine and stavudine, but not zidovudine (Kleim J-P, Rösner M,Winkler I, et al. (1995) J Acquir Immune Defic Syndr Suppl 3, 2).

Mutation at codon 181 has been reported to antagonize zidovudineresistance due to the typical 41 and 215 codon mutations, Zhang D,Caliendo A M, Eron J J, et al. (1994) Antimicrob Agents Chemother 38,282-287) suggesting that combination therapy with some NNRTIs andzidovudine may be feasible. Although an HIV mutant with tripleresistance to zidovudine, didanosine and nevirapine has been describedin vitro, (Larder B A, Kellam P, Kemp S D (1993) Nature 365, 451-453)treatment with this triple combination does provide superiorimmunological and virological responses to treatment with zidovudineplus didanosine alone over a 48-week period in patients with CD4 cellcounts <350/mm.

Combination therapy with zidovudine and the pyridinone derivativeL-697,661 prevents the appearance of the codon 181 mutation typicallyselected during monotherapy with this NNRTI, delaying the appearance ofhigh-level resistance to this compound. Changes in susceptibility tozidovudine were not examined in this study. (Staszewski S, Massari F E,Kober A, et al. (1995) J Infect Dis 171, 1159-1165). Concomitant oralternating zidovudine therapy does not delay the appearance ofresistance during nevirapine therapy; (Richman D D, Ibid) (Nunberg J H,Schleif W A, Boots E J, et al. (1990) J Virol 65, 4887-4892) (DeJong MD, Loewenthl M, Boucher C A B, et al. (1994) J Infect Dis 169,1346-1350) (Cheeseman S H, Havlir D, McLaughlin M M, et al. (1995) JAcquir Immune Defic Syndr 8, 141-151) however, the 181 mutant is notbeing observed during combination, the most common change being at codon190 (Richman D D, Ibid). This suggests that the codon 181 mutation whichis antagonistic to zidovudine resistance in vitro is not compatible, ornot preferred in vivo, selection favoring other mutations which allowfor reduced susceptibility to this NNRTI concomitant with zidovudineresistance.

The rapid development of reduced susceptibility to the NNRTIs suggestslimited utility of these agents, particularly as monotherapies, and hasled to the modification of these molecules in an attempt to delay theappearance of drug-resistant virus. A ‘second generation’ NNRTI, thepyridinone derivative L-702,019, demonstrated only a 3-fold change in ICbetween wild-type and codon 181 mutant HIV-1, and required multiplemutations to engender high-level resistance (Goldman M E, O'Brien J A,Ruffing T L, et al. (1993) Antimicrob Agents Chemother 37, 947-949).

It is an object of this invention to provide a drug susceptibility andresistance test capable of showing whether a viral population in apatient is resistant to a given prescribed drug. Another object of thisinvention is to provide a test that will enable the physician tosubstitute one or more drugs in a therapeutic regimen for a patient thathas become resistant to a given drug or drugs after a course of therapy.Yet another object of this invention is to provide a test that willenable selection of an effective drug regimen for the treatment of HIVinfections and/or AIDS. Yet another object of this invention is toprovide the means for identifying the drugs to which a patient hasbecome resistant, in particular identifying resistance to non-nucleosidereverse transcriptase inhibitors. Still another object of this inventionis to provide a test and methods for evaluating the biologicaleffectiveness of candidate drug compounds which act on specific viruses,viral genes and/or viral proteins particularly with respect to viraldrug resistance associated with non-nucleoside reverse transcriptaseinhibitors. It is also an object of this invention to provide the meansand compositions for evaluating HIV antiretroviral drug resistance andsusceptibility. This and other objects of this invention will beapparent from the specification as a whole.

SUMMARY OF THE INVENTION

The present invention relates to methods of monitoring, using phenotypicand genotypic methods, the clinical progression of humanimmunodeficiency virus infection and its response to antiviral therapy.The invention is also based, in part, on the discovery that geneticchanges in HIV reverse transcriptase (RT) which confer resistance toantiretroviral therapy may be rapidly determined directly from patientplasma HIV RNA using phenotypic or genotypic methods. The methodsutilize polymerase chain reaction (PCR) based assays. Alternatively,methods evaluating viral nucleic acid of viral protein in the absence ofan amplification step could utilize the teaching of this invention tomonitor and/or modify antiretroviral therapy. This invention is based inpart on the discovery of a mutation at codon 225 either alone or incombination with a mutation at codon 103 of HIV reverse transcriptase innon-nucleoside reverse transcriptase inhibitor (efavirenz) treatedpatient(s) in which the presence of the mutations correlates with anincrease in delavirdine susceptibility and little or no change innevirapine susceptibility. The mutations were found in plasma HIV RNAafter a period of time following initiation of therapy. The developmentof the mutant at codon 225 in addition to the mutation at codon 103 inHIV RT was found to be an indicator of the development of resistance andultimately of immunological decline. This invention is based in part onthe discovery of a mutation at codon 236 of RT was discovered to occurin non-nucleoside reverse transcriptase inhibitor (NNRTI) treatedpatients in which the presence of the mutation correlates with decreasedsusceptibility to delavirdine and no reduction in nevirapinesusceptibility. The development of the codon 190 and 103 and/or 101mutations in HIV RT was found to be an indicator of the development ofalterations in phenotypic susceptibility/resistance which has beenassociated with virologic failure and subsequent immunological decline.This invention is based in part on the discovery of a mutation at codon190 either alone or in combination with a mutation at codon 103 and/or101 of HIV reverse transcriptase in non-nucleoside reverse transcriptaseinhibitor (efavirenz) treated patient(s) in which the presence of themutations correlates with an increase in delavirdine susceptibility anda decrease in nevirapine susceptibility. The mutations were found inplasma HIV RNA after a period of time following initiation of NNRTItherapy. The development of the codon 236 and 103 and/or 181 mutationsin HIV RT was found to be an indicator of the development of alterationsin phenotypic susceptibility/resistance which has been associated withvirologic failure and subsequent immunological decline.

This invention is based in part on the discovery of a mutation at codon230 either alone or in combination with a mutation at codon 181 of HIVreverse transcriptase in non-nucleoside reverse transcriptase inhibitor(nevirapine) treated patient(s) in which the presence of the mutationscorrelates with a significant decrease in both delavirdine andnevirapine susceptibility. The mutations were found in plasma HIV RNAafter a period of time following initiation of NNRTI therapy. Thedevelopment of the codon 230 and 181 mutations in HIV RT was found to bean indicator of the development of alterations in phenotypicsusceptibility/resistance which has been associated with virologicfailure and subsequent immunological decline. This invention is based inpart on the discovery of a mutation at codon 181 of HIV reversetranscriptase in non-nucleoside reverse transcriptase inhibitor(nevirapine) treated patient(s) in which the presence of the mutationcorrelates with a moderate decrease in delavirdine susceptibility and asignificant decrease in nevirapine susceptibility and no change inefavirenz susceptibility. The mutation was found in plasma HIV RNA aftera period of time following initiation of NNRTI therapy. The developmentof the codon 181 mutation in HIV RT was found to be an indicator of thedevelopment of alterations in phenotypic susceptibility/resistance whichhas been associated with virologic failure and subsequent immunologicaldecline. This invention is based in part on the discovery of a mutationat codon 188 of HIV reverse transcriptase in non-nucleoside reversetranscriptase inhibitor (efavirenz) treated patient(s) in which thepresence of the mutation correlates with a slight decrease indelavirdine susceptibility and a substantial decrease in nevirapinesusceptibility. The mutation was found in plasma HIV RNA after a periodof time following initiation of NNRTI therapy. The development of thecodon 188 mutation in HIV RT was found to be an indicator of thedevelopment of alterations in phenotypic susceptibility/resistance whichhas been associated with virologic failure and subsequent immunologicaldecline. This invention is based in part on the discovery of a mutationat codon 188 of HIV reverse transcriptase in patient(s) with nopreviously reported exposure to non-nucleoside reverse transcriptaseinhibitors in which the presence of the mutations correlates with amoderate decrease in delavirdine susceptibility and a substantialdecrease in nevirapine susceptibility and a moderate decrease inefavirenz susceptibility. The mutation was found in plasma HIV RNA aftera period of time following initiation of anti-retroviral therapy. Thedevelopment of the codon 138 and 188 mutations in HIV RT was found to bean indicator of the development of alterations in phenotypicsusceptibility/resistance which has been associated with virologicfailure and subsequent immunological decline. This invention is based inpart on the discovery of a mutation at codon 98 of HIV reversetranscriptase in patient(s) with no previously reported exposure tonon-nucleoside reverse transcriptase inhibitors in which the presence ofthe mutation correlates with slight decrease in delavirdine, nevirapineand efavirenz susceptibility. The mutation was found in plasma HIV RNAafter a period of time following initiation of anti-retroviral therapy.The development of the codon 98 mutation in HIV RT was found to be anindicator of the development of alterations in phenotypicsusceptibility/resistance which has been associated with virologicfailure and subsequent immunological decline.

This invention is based in part on the discovery of a mutation at codon98 either alone or in combination with a mutation at codon 190 of HIVreverse transcriptase in patient(s) whose anti-retroviral treatment wasunknown in which the presence of the mutations correlates with anincrease in delavirdine susceptibility and substantial decrease in bothnevirapine and efavirenz susceptibility. The mutations were found inplasma HIV RNA. The development of the mutant at codon 98 in addition tothe mutation at codon 190 in HIV RT was found to be an indicator of thedevelopment of resistance and ultimately of immunological decline. Thisinvention is based in part on the discovery of a mutation at codon 181either alone or in combination with a mutation at codon 98 of HIVreverse transcriptase in non-nucleoside reverse transcriptase inhibitor(delavirdine) treated patient(s) in which the presence of the mutationscorrelates with a significant decrease in delavirdine susceptibility anda substantial decrease in efavirenz susceptibility. The mutations werefound in plasma HIV RNA after a period of time following initiation oftherapy. The development of the mutant at codon 98 in addition to themutation at codon 181 in HIV RT was found to be an indicator of thedevelopment of resistance and ultimately of immunological decline. Thisinvention is based in part on the discovery of a mutation at codon 101either alone or in combination with a mutation at codon 190, for example190s of HIV reverse transcriptase in non-nucleoside reversetranscriptase inhibitor (efavirenz) treated patient(s) in which thepresence of the mutations correlates with no change in delavirdinesusceptibility and a substantial decrease in both nevirapine andefavirenz susceptibility. The mutations were found in plasma HIV RNAafter a period of time following initiation of therapy. The developmentof the mutant at codon 101 in addition to the mutation at codon 190, forexample 190s in HIV RT was found to be an indicator of the developmentof resistance and ultimately of immunological decline. This invention isbased in part on the discovery of a mutation at codon 108 of HIV reversetranscriptase in patient(s) with no previously reported exposure tonon-nucleoside reverse transcriptase inhibitor in which the presence ofthe mutation correlates with no change in delavirdine susceptibility anda slight decrease in nevirapine susceptibility and no change inefavirenz susceptibility. The mutation was found in plasma HIV RNA aftera period of time following initiation of anti-retroviral therapy. Thedevelopment of the codon 108 mutation in HIV RT was found to be anindicator of the development of alterations in phenotypicsusceptibility/resistance which has been associated with virologicfailure and subsequent immunological decline.

This invention is based in part on the discovery of a mutation at codon101 either alone or in combination with a mutation at codon 103 and/or190 of HIV reverse transcriptase in patients with no previously reportedexposure to non-nucleoside reverse transcriptase inhibitors in which thepresence of the mutations correlates with changes in delavirdine,nevirapine and efavirenz susceptibility. Specifically, the presence ofmutations at 101 and 190, for example 190A, correlates with no change indelavirdine susceptibility and a substantial decrease in nevirapinesusceptibility and a significant decrease in efavirenz susceptibility.The presence of mutations at 103 and 190 correlates with a moderatedecrease in delavirdine susceptibility, a substantial decrease innevirapine susceptibility and a significant decrease in efavirenzsusceptibility. The mutations were found in plasma HIV RNA after aperiod of time following initiation of anti-retroviral therapy. Thedevelopment of the codon 101 and 103 and/or 190 mutations in HIV RT wasfound to be an indicator of the development of alterations in phenotypicsusceptibility/resistance which has been associated with virologicfailure and subsequent immunological decline. This invention is based inpart on the discovery of a mutation at codon 106 either alone or incombination with a mutation at codon 189 and/or 181 and 227 of HIVreverse transcriptase in non-nucleoside reverse transcriptase inhibitor(nevirapine) treated patient(s) in which the presence of the mutationscorrelates with changes in delavirdine, nevirapine and efavirenzsusceptibility. Specifically, the presence of mutations at 106 and 181correlates with a significant decrease in delavirdine susceptibility, asubstantial decrease in neviradine susceptibility and a slight decreasein efavirenz susceptibility. The presence of mutations at 106 and 189correlates with a slight decrease in delavirdine susceptibility, amoderate decrease in nevirapine susceptibility and no change inefavirenz susceptibility. The presence of mutations at 106 and 227correlates with a slight decrease in delavirdine susceptibility, asubstantial decrease in nevirapine susceptibility and a slight decreasein efavirenz susceptibility. The presence of mutations at 181 and 227correlates with an increase in delavirdine susceptibility, a significantdecrease in nevirapine susceptibility and an increase in efavirenzsusceptibility. The presence of mutations at 106 and 181 and 227correlates with a moderate decrease in delavirdine susceptibility, asubstantial decrease in nevirapine susceptibility and a slight decreasein efavirenz susceptibility. The mutations were found in plasma HIV RNAafter a period of time following initiation of NNRTI therapy. Thedevelopment of the codon 106 and 189 and/or 181 and 227 mutations in HIVRT was found to be an indicator of the development of alterations inphenotypic susceptibility/resistance which has been associated withvirologic failure and subsequent immunological decline. This inventionis based in part on the discovery of a mutation at codon 103 eitheralone or in combination with a mutation at codon 100 and/or 188 of HIVreverse transcriptase in non-nucleoside reverse transcriptase inhibitor(nevirapine) created patient(s) in which the presence of the mutationscorrelates with changes in delavirdine, nevirapine and efavirenzsusceptibility. Specifically, the presence of mutations at 103 and 188correlates with a substantial decrease in delavirdine susceptibility, asubstantial decrease in nevirapine susceptibility and a substantialdecrease in efavirenz susceptibility. The presence of mutations at 100and 103 correlates with a substantial decrease in delavirdinesusceptibility, a moderate decrease in nevirapine susceptibility and asubstantial decrease in efavirenz susceptibility. The presence ofmutations at 103 and 100 and 188 correlates with a substantial decreasein delavirdine susceptibility, a substantial decrease in nevirapinesusceptibility and a substantial decrease in efavirenz susceptibility.The mutations were found in plasma HIV RNA after a period of timefollowing initiation of NNRTI therapy. The development of the codon 103and 100 and/or 188 mutations in HIV RT was found to be an indicator ofthe development of alterations in phenotypic susceptibility/resistancewhich has been associated with virologic failure and subsequentimmunological decline.

In a further embodiment of the invention, PCR based assays, includingphenotypic and genotypic assays, may be used to detect mutations atcodon 225 in combination with mutations at other codons including 103 ofHIV RT which correlate with a specific pattern of resistance toantiretroviral therapies and subsequent immunologic decline. In yetanother embodiment of the invention, PCR based assays, includingphenotypic and genotypic assays, may be used to detect mutations atcodon 236 either alone or in combination with mutations at other codonsincluding 103 and/or 181 of HIV RT which correlate with resistance toantiretroviral therapy and immunologic decline. In yet anotherembodiment of the invention, PCR based assays, including phenotypic andgenotypic assays, may be used to detect mutations at codon 190 (G190S)either alone or in combination with mutation at codon 101 (K101E) of HIVRT which correlates with resistance to antiretroviral therapy andimmunologic decline. In still another embodiment of the invention, PCRbased assays, including phenotypic and genotypic assays, may be used todetect mutations at codon 190 (G190A) either alone or in combinationwith mutation at codon 103 (K103N) of HIV RT which correlates withresistance to antiretroviral therapy and immunologic decline. In yetanother embodiment of the invention, PCR based assays, includingphanotypic and genotypic assays, may be used to detect mutations atcodon 230 either alone or in combination with mutation at codon 181 ofHIV RT which correlates with resistance to antiretroviral therapy andimmunologic decline. In yet another embodiment of the invention, PCRbased assays, including phenotypic and genotypic assays, may be used todetect a mutation at codon 181 of HIV RT which correlates withresistance to antiretroviral therapy and immunologic decline. In yetanother embodiment of the invention, PCR based assays, includingphenotypic and genotypic assays, may be used to detect a mutation atcodon 188 of HIV RT which correlates with resistance to antiretroviraltherapy and immunologic decline. In yet another embodiment of theinvention, PCR based assays, including phenotypic and genotypic assays,may be used to detect mutations at codon 138 either alone or incombination with mutation at codon 188 of HIV RT which correlates withresistance to antiretroviral therapy and immunologic decline. In yetanother embodiment of the invention, PCR based assays, includingphenotypic and genotypic assays, may be used to detect a mutation atcodon 98 of HIV RT which correlates with resistance to antiretroviraltherapy and immunologic decline. In yet another embodiment of theinvention, PCR based assays, including phenotypic and genotypic assays,may be used to detect mutations at codon 98 either alone or incombination with mutation at codon 190 of HIV RT which correlates withresistance to antiretroviral therapy and immunologic decline. In yetanother embodiment of the invention, PCR based assays, includingphenotypic and genotypic assays, may be used to detect mutations atcodon 181 either alone or in combination with mutation at codon 98 ofHIV RT which correlates with resistance to antiretroviral therapy andimmunologic decline. In yet another embodiment of the invention, PCRbased assays, including phenotypic and genotypic assays, may be used todetect mutations at codon 101 either alone or in combination withmutation at codon 190, for example 190s of HIV RT which correlates withresistance to antiretroviral therapy and immunologic decline. In yetanother embodiment of the invention, PCR based assays, includingphenotypic and genotypic assays, may be used to detect a mutation atcodon 108 of HIV RT which correlates with resistance to antiretroviraltherapy and immunologic decline. In yet another embodiment of theinvention, PCR based assays, including phenotypic and genotypic assays,may be used to detect mutations at codon 101 either alone or incombination with mutations at codon 103 and/or 190 of HIV RT whichcorrelates with resistance to antiretroviral therapy and immunologicdecline. In yet another embodiment of the invention, PCR based assays,including phenotypic and genotypic assays, may be used to detectmutations at codon 106 either alone or in combination with mutations atcodon 189 and/or 181 and 227 of HIV RT which correlates with resistanceto antiretroviral therapy and immunologic decline. In yet anotherembodiment of the invention, PCR based assays, including phenotypic andgenotypic assays, may be used to detect mutations at codon 188 eitheralone or in combination with mutation at codon 100 and/or 103 of HIV RTwhich correlates with resistance to antiretroviral therapy andimmunologic decline. Once mutations at codon 225 and 103 have beendetected in a patient undergoing NNRTI antiretroviral therapy, analteration in the therapeutic regimen must be considered. Similarly,once mutations at codon 236 and/or 103 and/or 181 have been detected ina patient undergoing certain NNRTI antiretroviral therapy, an alterationin the therapeutic regimen must be considered. Similarly, once mutationsat codon 190 and/or 103 and/or 101 have been detected in a patientundergoing certain NNRTI antiretroviral therapy, an alteration in thetherapeutic regimen must be considered. Similarly, once mutations atcodon 230 and/or 181 have been detected in a patient undergoing certainNNRTI antiretroviral therapy, an alteration in the therapeutic regimenmust be considered. Similarly, once a mutation at codon 181 has beendetected in a patient undergoing certain NNRTI antiretroviral therapy,an alteration in the therapeutic regimen must be considered. Similarly,once a mutation at codon 188 has been detected in a patient undergoingcertain NNRTI antiretroviral therapy, an alteration in the therapeuticregimen must be considered. Similarly, once mutations at codon 13 8and/or 188 have been detected in a patient undergoing certain NNRTIantiretroviral therapy, an alteration in the therapeutic regimen must beconsidered. Similarly, once a mutation at codon 98 has been detected ina patient undergoing certain NNRTI antiretroviral therapy, an alterationin the therapeutic regimen must be considered. Similarly, once mutationsat codon 98 and/or 190 have been detected in a patient undergoingcertain NNRTI antiretroviral therapy, an alteration in the therapeuticregimen must be considered. Similarly, once mutations at codon 181and/or 98 have been detected in a patient undergoing certain NNRTIantiretroviral therapy, an alteration in the therapeutic regimen must beconsidered. Similarly, once mutations at codon 101 and/or 190, forexample 190S, have been detected in a patient undergoing certain NNRTIantiretroviral therapy, an alteration in the therapeutic regimen must beconsidered. Similarly, once a mutation at codon 108 has been detected ma patient undergoing certain NNRTI antiretroviral therapy, an alterationin the therapeutic regimen must be considered. Similarly, once mutationsat codon 101 and/or 103 and/or 190, for example 190A, have been detectedin a patient undergoing certain NNRTI antiretroviral therapy, analteration in the therapeutic regimen must be considered. Similarly,once mutations at codon 106 and/or 189 and/or 181 and/or 227 have beendetected in a patient undergoing certain NNRTI antiretroviral therapy,an alteration in the therapeutic regimen must be considered. Similarly,once mutations at codon 188 and/or 100 and/or 103 have been detected ina patient undergoing certain NNRTI antiretroviral therapy, an alterationin the therapeutic regimen must be considered. The timing at which amodification of the therapeutic regimen should be made, following theassessment of the antiretroviral therapy using PCR based assays, maydepend on several factors including the patient's viral load, CD4 count,and prior treatment history.

In another aspect of the invention there is provided a method forassessing the effectiveness of a non-nucleoside reverse transcriptaseantiretroviral drug comprising: (a) introducing a resistance test vectorcomprising a patient-derived segment and an indicator gene into a hostcell; (b) culturing the host cell from step (a); (c) measuringexpression of the indicator gene in a target host cell whereinexpression of the indicator gene is dependent upon the patient-derivedsegment; and (d) comparing the expression of the indicator gene fromstep (c) with the expression of the indicator gene measured when steps(a) (c) are carried out in the absence of the NNRTI anti-HIV drug,wherein a test concentration of the NNRTI, anti-HIV drug is presented atsteps (a) (c); at steps (b) (c); or at step (c).

This invention also provides a method for assessing the effectiveness ofnon-nucleoside reverse transcriptase antiretroviral therapy in a patientcomprising: (a) developing a standard curve of drug susceptibility foran NNRTI anti-HIV drug; (b) determining NNRTI anti-HIV drugsusceptibility in the patient using the susceptibility test describedabove; and (c) comparing the NNRTI anti-HIV drug susceptibility in step(b) with the standard curve determined in step (a), wherein a decreasein NNRTI anti-HIV susceptibility indicates development of anti-HIV drugresistance in the patient.

This invention also provides a method for evaluating the biologicaleffectiveness of a candidate HIV antiretroviral drug compoundcomprising: (a) introducing a resistance test vector comprising apatient-derived segment and an indicator gene into a host cell; (b)culturing the host cell from step (a); (c) measuring expression of theindicator gene in a target host cell wherein expression of the indicatorgene is dependent upon the patient-derived segment; and (d) comparingthe expression of the indicator gene from step (c) with the expressionof the indicator gene measured when steps (a) (c) are carried out in theabsence of the candidate anti-viral drug compound, wherein a testconcentration of the candidate anti-viral drug compound is present atsteps (a) (c); at steps (b) (c); or at step (c).

The expression of the indicator gene in the resistance test vector inthe target cell is ultimately dependent upon the action of thepatient-derived segment sequences. The indicator gene may be functionalor non-functional.

In another aspect this invention is directed to antiretroviral drugsusceptibility and resistance tests for HIV/AIDS. Particular resistancetest vectors of the invention for use in the HIV/AIDS antiretroviraldrug susceptibility and resistance test are identified.

In yet another aspect this invention provides for the identification andassessment of the biological effectiveness of potential therapeuticantiretroviral compounds for the treatment of HIV and/or AIDS. Inanother aspect, the invention is directed to a novel resistance testvector comprising a patient-derived segment further comprising one ormore mutations on the RT gene and an indicator gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

Resistance Test Vector. A diagrammatic representation of the resistancetest vector comprising a patient derived segment and an indicator gene.

FIG. 2

Two Cell Assay. Schematic Representation of the Assay. A resistance testvector is generated by cloning the patient-derived segment into anindicator gene viral vector. The resistance test vector is thenco-transfected with an expression vector that produces amphotropicmurine leukemia virus (MLV) envelope protein or other viral or cellularproteins which enable infection. Pseudotyped viral particles areproduced containing the protease (PR) and the reverse transcriptase (RT)gene products encoded by the patient-derived sequences. The particlesare then harvested and used to infect fresh cells. Using defective PRand RT sequences it was shown that luciferase activity is dependent onfunctional PR and RT. PR inhibitors are added to the cells followingtransfection and are thus present during particle maturation. RTinhibitors, on the other hand, are added to the cells at the time of orprior to viral particle infection. The assay is performed in the absenceof drug and in the presence of drug over a wide range of concentrations.The amount of luciferase is determined and the percentage (%) inhibitionis calculated at the different drug concentrations tested.

FIG. 3

Examples of phenotypic drug susceptibility profiles. Data are analyzedby plotting the percent inhibition of luciferase activity vs. logconcentration. This plot is used to calculate the drug concentrationthat is required to inhibit virus replication by 50% (IC₅₀) or by 95%(IC₉₅). Shifts in the inhibition curves towards higher drugconcentrations are interpreted as evidence of drug resistance. Threetypical curves for a nucleoside reverse transcriptase inhibitor (AZT), anon-nucleoside reverse transcriptase inhibitor (delavirdine), and aprotease inhibitor (ritonavir) are shown. A reduction in drugsusceptibility (resistance) is reflected in a shift in the drugsusceptibility curve toward higher drug concentrations (to the right) ascompared to a baseline (pre-treatment) sample or a drug susceptiblevirus control, such as PNL4-3 or HXB-2, when a baseline sample is notavailable.

FIG. 4

Phenotypic drug susceptibility and resistance profile: patient 487. APCR-based phenotypic susceptibility assay was carried out giving thephenotypic drug susceptibility and resistance profile showing increasedresistance to both delavirdine and nevirapine. This is an example of thefirst pattern of NNRTI susceptibility/resistance. Evaluation of thisvirus from plasma showed HIV reverse transcriptase having mutations atcodons 184 (M184V) associated with 3TC resistance and at 103 (K103N)associated with both delavirdine and nevirapine resistance.

FIG. 5

Phenotypic drug susceptibility and resistance profile of site directedreverse transcriptase mutants. A PCR-based phenotypic susceptibilityassay was carried out giving the phenotypic drug susceptibility andresistance profile for site directed mutants having mutations at codons103 and 181 (K103N; Y181C) demonstrating resistance to both delavirdineand nevirapine. The double mutant demonstrates the additive effect ofboth mutations resulting in a further increase in resistance.

FIG. 6

Phenotypic drug susceptibility and resistance profile: Patient 268. APCR-based phenotypic susceptibility assay was carried out giving thephenotypic drug susceptibility and resistance profile showing theevaluation of virus from plasma with HIV reverse transcriptase havingphenotypic resistance to delavirdine but not nevirapine. This is anexample of the second pattern of NNRTI susceptibility/resistance. Thispatient virus is resistant to all of the protease inhibitors tested andalso has significant resistance to AZT and 3TC and shows slight shiftsin susceptibility to ddC, ddI, and d4T. Evaluation of this virus fromplasma using a PCR and sequencing based genotypic assay showed HIVreverse transcriptase having mutations an codons 103 and 236 (K103N;P236L). The P236L mutation was previously reported to cause delavirdineresistance and nevirapine hypersensitivity (Dueweke T J et al. (1993)Proc Natl Acad Sci 90, 4713-4717). However, in this patient sample,while there was delavirdine resistance nevirapine susceptibility was thesame as wild type.

FIG. 7

Phenotypic drug susceptibility and resistance profile of site-directedreverse transcriptase mutant (P236L). A PCR-based phenotypicsusceptibility assay was carried out giving the phenotypic drugsusceptibility and resistance profile showing the susceptibility todelavirdine and nevirapine of the P236L site-directed mutagenesismutant. This result is identical to that observed in the patient virussample shown in FIG. 6. The next two panels show the K103N site-directedmutagenesis mutant and the two panels below show the double mutantK103N+P236L. The P236L mutation is additive to the K103N causing severeresistance to delavirdine while having no effect on nevirapineresistance due to K1C3N. The right side of the figure shows a similarresult when the P236L mutation is added to the Y181→C mutation.

FIG. 8A

Phenotypic Drug Susceptibility and Resistance Profile: Patients 302.This is one example of the third pattern of NNRTIsusceptibility/resistance. Phenotypic analysis of the patient virusdemonstrated reduced susceptibility to both delavirdine and nevirapine.This pattern is characterized by a larger reduction of nevirapinesusceptibility compared to the reduction of delavirdine susceptibility.Genotypic analysis of the patient virus demonstrated the presence of theRT mutations K103N associated with nevirapine and delavirdine resistanceand P225H.

FIG. 8B

Phenotypic Drug Susceptibility and Resistance Profile: Patients 780.This is a second example of the third pattern of NNRTIsusceptibility/resistance. Phenotypic analysis of the patient virusdemonstrated reduced susceptibility to both delavirdine and nevirapine.This pattern is characterized by a larger reduction of nevirapinesusceptibility compared to the reduction of delavirdine susceptibility.Genotypic analysis of the patient virus demonstrated the presence of theRT mutations K103N associated with nevirapine and delavirdine resistanceand P225H.

FIG. 8C

Phenotypic Drug Susceptibility and Resistance Profile: Individual VirusClones of Patient 302. Genotypic analysis of individual virus clonesfrom patient 302 revealed viruses containing the K103N mutation withoutthe P225H mutation (K103N, I135M, R211K) and viruses containing theK103N mutation with the P225H mutation (K103N, P225H). Phenotypiccharacterization of these virus clones indicates that the P225H mutationreduces the amount delavirdine resistance associated with the K103Nmutation (compare bottom panels), but does not alter the amount ofnevirapine resistance associated with the K103N mutation (compare toppanels).

FIG. 8D

Phenotypic Drug Susceptibility and Resistance Profile: Site DirectedReverse Transcriptase Mutants. Phenotypic characterization of a viruscontaining the site directed RT mutation P225H indicates that thismutation increases susceptibility to delavirdine, but not nevirapine(compare top panels). Phenotypic characterization of a virus containingthe site directed RT mutations P225H plus K103N or P225H plus Y181Cindicate that the P225H mutation decreases the amount of delavirdineresistance associated with either K103N or Y181C, but does not decreasethe amount of nevirapine resistance associated with K103N or Y181C(compare corresponding middle and bottom panels).

FIG. 9A

Phenotypic Drug Susceptibility and Resistance Profile: Patients 644.This is one example of the fourth pattern of NNRTI susceptibility andresistance. Phenotypic analysis of the patient virus demonstrated by alarge reduction in susceptibility to nevirapine, but not delavirdine.Genotypic analysis of the patient virus demonstrated the presence of theRT mutations G190S, as well as the K101E mutation associated withreductions in susceptibility to atevirdine, DMP266, L-697,661 andUC-10,38,57 (Schinazi, Mellors, Larder resistance table).

FIG. 9B

Phenotypic Drug Susceptibility and Resistance Profile: Site DirectedReverse Transcriptase Mutants. Phenotypic characterizations of virusescontaining either site directed RT mutations G190A, or G190S indicatethat these mutations greatly reduce susceptibility to nevirapine, andslightly increase susceptibility to delavirdine (compare top panels).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of monitoring the clinicalprogression of HIV infection in patients receiving antiretroviraltherapy, particularly non-nucleoside reverse transcriptase inhibitorantiretroviral therapy.

In one embodiment, the present invention provides for a method ofassessing the effectiveness of antiretroviral therapy of a patientcomprising (i) collecting a biological sample from an HIV-infectedpatient; and (ii) determining whether the biological sample comprisesnucleic acid encoding HIV RT having a mutation at one or more positionsin the RT. The mutation(s) correlate positively with alterations inphenotypic susceptibility/resistance. In a specific embodiment, theinvention provides for a method of assessing the effectiveness of NNRTIantiretroviral therapy of a patient comprising (i) collecting abiological sample from an HIV-infected patient; and (ii) determiningwhether the biological sample comprises nucleic acid encoding HIV RThaving a mutation at codon 225 and 103. This invention established,using a phenotypic susceptibility assay, that mutations at codon 225either alone or in combination with a mutation at codon 103 of HIVreverse transcriptase are correlated with an increase in delavirdinesusceptibility, little or no change in nevirapine susceptibility andlittle or no change in efavirenz susceptibility. In another specificembodiment, the invention provides for a method of evaluating theeffectiveness of NNRTI antiretroviral therapy of a patient comprising(i) collecting a biological sample from an HIV-infected patient; and(ii) determining whether the biological sample comprises nucleic acidencoding HIV RT having a mutation at codon(s) 236 and 103 and/or 181.This invention established, using a phenotypic susceptibility assay,that mutations at codon 236 either alone or in combination with amutation at codon 103 and/or 181 of HIV reverse transcriptase arecorrelated with a decrease in delavirdine susceptibility (increasedresistance) and no change in nevirapine susceptibility. The 236 mutationalone or on a Y181C background has no effect on efavirenz susceptibilitybut restores a significant portion of the loss of susceptibility causedby a 103N mutation. In another specific embodiment, the inventionprovides for a method of evaluating the effectiveness of NNRTIantiretroviral therapy of a patient comprising (i) collecting abiological sample from an HIV-infected patient; and (ii) determiningwhether the biological sample comprises nucleic acid encoding HIV RThaving a mutation at codon(s) 230 and/or 181. This inventionestablished, using a phenotypic susceptibility assay, that mutations atcodon 230 either alone or in combination with a mutation at codon 181 ofHIV reverse transcriptase are correlated with a significant decrease indelavirdine susceptibility (increased resistance), significant decreasein nevirapine susceptibility. In another specific embodiment, theinvention provides for a method of evaluating the effectiveness of NNRTIantiretroviral therapy of a patient comprising (i) collecting abiological sample from an HIV-infected patient; and (ii) determiningwhether the biological sample comprises nucleic acid encoding HIV RThaving a mutation at codon 181. This invention established, using aphenotypic susceptibility assay, that a mutation at codon 181 of HIVreverse transcriptase is correlated with a moderate decrease indelavirdine susceptibility (increased resistance), significant decreasein nevirapine susceptibility and no change in efavirenz susceptibility.In another specific embodiment, the invention provides for a method ofevaluating the effectiveness of NNRTI antiretroviral therapy of apatient comprising (i) collecting a biological sample from anHIV-infected patient; and (ii) determining whether the biological samplecomprises nucleic acid encoding HIV RT having a mutation at codon 188.This invention established, using a phenotypic susceptibility assay,that a mutation at codon 188 of HIV reverse transcriptase are correlatedwith a slight decrease in delavirdine susceptibility (increasedresistance), a substantial decrease in nevirapine susceptibility and asignificant decrease in efavirenz susceptibility. In another specificembodiment, the invention provides for a method of evaluating theeffectiveness of NNRTI antiretroviral therapy of a patient comprising(i) collecting a biological sample from an HIV-infected patient; and(ii) determining whether the biological sample comprises nucleic acidencoding HIV RT having a mutation at codon(s) 138 and/or 188. Thisinvention established, using a phenotypic susceptibility assay, thatmutations at codon 138 either alone or in combination with a mutation atcodon 188 of HIV reverse transcriptase are correlated with a moderatedecrease in delavirdine susceptibility (increased resistance), asubstantial decrease in nevirapine susceptibility and a moderatedecrease in efavirenz susceptibility. In another specific embodiment,the invention provides for a method of evaluating the effectiveness ofNNRTI antiretroviral therapy of a patient comprising (i) collecting abiological sample from an HIV-infected patient; and (ii) determiningwhether the biological sample comprises nucleic acid encoding HIV RThaving a mutation at codon(s) 98. This invention established, using aphenotypic susceptibility assay, that mutations at codon 98 of HIVreverse transcriptase are correlated with a slight decrease indelavirdine susceptibility (increased resistance), a slight decrease innevirapine susceptibility and a slight decrease in efavirenzsusceptibility. In another specific embodiment, the invention providesfor a method of evaluating the effectiveness of NNRTI antiretroviraltherapy of a patient comprising (i) collecting a biological sample froman HIV-infected patient; and (ii) determining whether the biologicalsample comprises nucleic acid encoding HIV RT having a mutation atcodon(s) 98 and/or 190. This invention established, using a phenotypicsusceptibility assay, that mutations at codon 98 either alone or incombination with a mutation at codon 190 of HIV reverse transcriptaseare correlated with an increase in delavirdine susceptibility (decreasedresistance), a substantial decrease in nevirapine susceptibility and asubstantial decrease in efavirenz susceptibility. In another specificembodiment, the invention provides for a method of evaluating theeffectiveness of NNRTI antiretroviral therapy of a patient comprising(i) collecting a biological sample from an HIV-infected patient; and(ii) determining whether the biological sample comprises nucleic acidencoding HIV RT having a mutation at codon(s) 181 and/or 98. Thisinvention established, using a phenotypic susceptibility assay, thatmutations at codon 181 either alone or in combination with a mutation atcodon 98 of HIV reverse transcriptase are correlated with a significantdecrease in delavirdine susceptibility (increased resistance), asubstantial decrease in nevirapine susceptibility and a slight decreasein efavirenz susceptibility. In another specific embodiment, theinvention provides for a method of evaluating the effectiveness of NNRTIantiretroviral therapy of a patient comprising (i) collecting abiological sample from an HIV-infected patient; and (ii) determiningwhether the biological sample comprises nucleic acid encoding HIV RThaving a mutation at codon(s) 101 and/or 190, for example L90S. Thisinvention established, using a phenotypic susceptibility assay, thatmutations at codon 101 either alone or in combination with a mutation atcodon 190 of HIV reverse transcriptase are correlated with no change indelavirdine susceptibility (wild-type), a substantial decrease innevirapine susceptibility and a substantial decrease in efavirenzsusceptibility. In another specific embodiment, the invention providesfor a method of evaluating the effectiveness of NNRTI antiretroviraltherapy of a patient comprising (i) collecting a biological sample froman HIV-infected patient; and (ii) determining whether the biologicalsample comprises nucleic acid encoding HIV RT having a mutation atcodon(s) 108. This invention established, using a phenotypicsusceptibility assay, that a mutation at codon 108 of HIV reversetranscriptase are correlated with no change in delavirdinesusceptibility (wild-type), a slight decrease in nevirapinesusceptibility and no change in efavirenz susceptibility. In anotherspecific embodiment, the invention provides for a method of evaluatingthe effectiveness of NNRTI antiretroviral therapy of a patientcomprising (i) collecting a biological sample

from an HIV-infected patient; and (ii) determining whether thebiological sample comprises nucleic acid encoding HIV RT having amutation at codon(s) 101 and 103 and/or 190. This invention established,using a phenotypic susceptibility assay, that mutations at codon 101either alone or in combination with a mutation at codon 103 and/or 190of HIV reverse transcriptase are correlated with a either no change (101and 190) or a moderate decrease (103 and 190, for example 190A) indelavirdine susceptibility (increased resistance), a substantialdecrease in nevirapine susceptibility and a significant decrease inefavirenz susceptibility. In another specific embodiment, the inventionprovides for a method of evaluating the effectiveness of NNRTIantiretroviral therapy of a patient comprising (i) collecting abiological sample from an HIV-infected patient; and (ii) determiningwhether the biological sample comprises nucleic acid encoding HIV RThaving a mutation at codon(s) 106 and/or 189 and/or 181 and/or 227. Thisinvention established, using a phenotypic susceptibility assay, thatmutations at codon 106 either alone oar in combination with a mutationat codon 189 and/or 181 and/or 227 of HIV reverse transcriptase arecorrelated with changes in delavirdine, nevirapine and efavirenzsusceptibility. Specifically, the presence of mutations at 106 and 181correlates with a significant decrease in delavirdine susceptibility, asubstantial decrease in nevirapine susceptibility and a slight decreasein efavirenz susceptibility. The presence of mutations at 106 and 189correlates with a slight decrease in delavirdine susceptibility, amoderate decrease in nevirapine susceptibility and no change inefavirenz susceptibility. The presence of mutations at 106 and 227correlates with a slight decrease in delavirdine susceptibility, asubstantial decrease in nevirapine susceptibility and a slight decreasein efavirenz susceptibility. The presence of mutations at 181 and 227correlates with an increase in delavirdine susceptibility, a significantdecrease in nevirapine susceptibility and an increase in efavirenzsusceptibility. The presence of mutations at 106 and 181 and 227correlates with a moderate decrease in delavirdine susceptibility, asubstantial decrease in nevirapine susceptibility and a slight decreasein efavirenz susceptibility. In another specific embodiment, theinvention provides for a method of evaluating the effectiveness of NNRTIantiretroviral therapy of a patient comprising (i) collecting abiological sample from an HIV-infected patient; and (ii) determiningwhether the biological sample comprises nucleic acid encoding HIV RThaving a mutation at codon(s) 188 and 100 and/or 103. This inventionestablished, using a phenotypic susceptibility assay, that mutations atcodon 188 either alone or in combination with a mutation at codon 100and/or 103 of HIV reverse transcriptase are correlated changes indelavirdine, nevirapine and efavirenz susceptibility. Specifically, thepresence of mutations at 103 and 188 correlates with a substantialdecrease in delavirdine susceptibility, a substantial decrease innevirapine susceptibility and a substantial decrease in efavirenzsusceptibility. The presence of mutations at 100 and 103 correlates witha substantial decrease in delavirdine susceptibility, a moderatedecrease in nevirapine susceptibility and a substantial decrease inefavirenz susceptibility. The presence of mutations at 103 and 100 and188 correlates with a substantial decrease in delavirdinesusceptibility, a substantial decrease in nevirapine susceptibility anda substantial decrease in efavirenz susceptibility. Under the foregoingcircumstances, the phenotypic susceptibility/resistance profile andgenotypic profile of the HIV virus infecting the patient has beenaltered reflecting some change in the response to the antiretroviralagent. In the case of NNRTI antiretroviral therapy, the HIV virusinfecting the patient may be resistant to one or more but not another ofthe NNRTIs as described herein. It therefore may be desirable afterdetecting the mutation, to either increase the dosage of theantiretroviral agent, change to another antiretroviral agent, or add oneor more additional antiretroviral agents to the patient's therapeuticregimen. For example, if the patient was being treated with efavirenz(DMP-266) when the 225 mutation arose, the patient's therapeutic regimenmay desirably be altered by either (i) changing to a different NNRTIantiretroviral agent, such as delavirdine or nevirapine and stoppingefavirenz treatment; or (ii) increasing the dosage of efavirenz; or(iii) adding another antiretroviral agent to the patient's therapeuticregimen. The effectiveness of the modification in therapy may beevaluated by monitoring viral burden such as by HIV RNA copy number. Adecrease in HIV RNA copy number correlates positively with theeffectiveness of a treatment regimen.

The phrase “correlates positively,” as used herein, indicates that aparticular result renders a particular conclusion more likely than otherconclusions.

Another preferred, non-limiting, specific embodiment of the invention isas follows: A method of assessing the effectiveness of NNRTI therapy ofa patient comprising (i) collecting a biological sample from anHIV-infected patient; (ii) amplifying the HIV-encoding RNA in thebiological sample by converting the RNA to cDNA and amplifying HIVsequences using HIV primers that result in a PCR product that comprisesthe RT gene; (iii) performing PCR using primers that result in PCRproducts comprising wild type or mutant 225 and 103 codons; and (iv)determining, via the products of PCR, the presence or absence of amutation at codon 225 or 103 or both. Yet another preferred,non-limiting specific embodiment of the invention is as follows: Amethod of assessing the effectiveness of NNRTI therapy of a patientcomprising (i) collecting a plasma sample from an HIV-infected patient;(ii) amplifying the HIV-encoding RNA in the plasma sample by convertingthe RNA to cDNA and amplifying HIV sequences using HIV primers thatresult in a PCR product that comprises the RT gene; (iii) performing PCRusing primers that result in PCR products comprising the wild type ormutations at codons 103 and/or 181 and 236; and (iv) determining, viathe products of PCR, the presence or absence of a mutation at codon 236and 103 and/or 181. Yet another preferred, non-limiting specificembodiment of the invention is as follows: A method of assessing theeffectiveness of NNRTI therapy of a patient comprising (i) collecting aplasma sample from an HIV-infected patient; (ii) amplifying theHIV-encoding RNA in the plasma sample by converting the RNA to cDNA andamplifying HIV sequences using HIV primers that result in a PCR productthat comprises the RT gene; (iii) performing PCR using primers thatresult in PCR products comprising the wild type or mutations at codon101 and 190 (G190S); and (iv) determining, via the products of PCR, thepresence or absence of a mutation at codon 190 (G190S) and 103. Yetanother preferred, non-limiting specific embodiment, of the invention isas follows: A method of assessing the effectiveness of NNRTI therapy ofa patient comprising (i) collecting a plasma sample from an HIV-infectedpatient; (ii) amplifying the HIV-encoding RNA in the plasma sample byconverting the RNA to cDNA and amplifying HIV sequences using HIVprimers that result in a PCR product that comprises the RT gene; (iii)performing PCR using primers that result in PCR products comprising thewild type or mutations at codon 103 and 190 (G190A); and (iv)determining, via the products of PCR, the presence or absence of amutation at codon 190 (G190A) and 103. Yet another preferred,non-limiting specific embodiment of the invention is as follows: Amethod of assessing the effectiveness of NNRTI therapy of a patientcomprising (i) collecting a plasma sample from an HIV-infected patient;(ii) amplifying the HIV-encoding RNA in the plasma sample by convertingthe RNA to cDNA and amplifying HIV sequences using HIV primers thatresult in a PCR product that comprises the RT gene; (iii) performing PCRusing primers that result in PCR products comprising the wild type ormutations at codon 230 and 181; and (iv) determining, via the productsof PCR, the presence or absence of a mutation at codon 230 and 181. Yetanother preferred, non-limiting specific embodiment of the invention isas follows: A method of assessing the effectiveness of NNRTI therapy ofa patient comprising (i) collecting a plasma sample from an HIV-infectedpatient; (ii) amplifying the HIV-encoding RNA in the plasma sample byconverting the RNA to cDNA and amplifying HIV sequences using HIVprimers that result in a PCR product that comprises the RT gene; (iii)performing PCR using primers that result in PCR products comprising thewild type or mutation at 181; and (iv) determining, via the products ofPCR, the presence or absence of a mutation at codon 181. Yet anotherpreferred, non-limiting specific embodiment of the invention is asfollows: A method of assessing the effectiveness of NNRTI therapy of apatient comprising (i) collecting a plasma sample from an HIV-infectedpatient; (ii) amplifying the HIV-encoding RNA in the plasma sample byconverting the RNA to cDNA and amplifying HIV sequences using HIVprimers that result in a PCR product that comprises the RT gene; (iii)performing PCR using primers that result in PCR products comprising thewild type or mutation at codon 188; and (iv) determining, via theproducts of PCR, the presence or absence of a mutation at codon 188. Yetanother preferred, non-limiting specific embodiment of the invention isas follows: A method of assessing the effectiveness of NNRTI therapy ofa patient comprising (i) collecting a plasma sample from an HIV-infectedpatient; (ii) amplifying the HIV-encoding RNA in the plasma sample byconverting the RNA to cDNA and amplifying HIV sequences using HIVprimers that result in a PCR product that comprises the RT gene; (iii)performing PCR using primers that result in PCR products comprising thewild type or mutations at codon 138 and 188; and (iv) determining, viathe products of PCR, the presence or absence of a mutation at codon 138and 188. Yet another preferred, non-limiting specific embodiment, of theinvention is as follows: A method of assessing the effectiveness ofNNRTI therapy of a patient comprising (i) collecting a plasma samplefrom an HIV-infected patient; (ii) amplifying the HIV-encoding RNA inthe plasma sample by converting the RNA to cDNA and amplifying HIVsequences using HIV primers that result in a PCR product that comprisesthe RT gene; (iii) performing PCR using primers that result in PCRproducts comprising the wild type or mutation at codon 98 and (iv)determining, via the products of PCR, the presence or absence of amutation at codon 98. Yet another preferred, non-limiting specificembodiment of the invention is as follows: A method of assessing theeffectiveness of NNRTI therapy of a patient comprising (i) collecting aplasma sample from an HIV-infected patient; (ii) amplifying theHIV-encoding RNA in the plasma sampled by converting the RNA to cDNA andamplifying HIV sequences using HIV primers that result in a PCR productthat comprises the RT gene; (iii) performing PCR using primers thatresult in PCR products comprising the wild type or mutations at codon 98and 190; and (iv) determining, via the products of PCR, the presence orabsence of a mutation at codon 190 and 98. Yet another preferred,non-limiting specific embodiment of the invention is as follows: Amethod of assessing the effectiveness of NNRTI therapy of a patientcomprising (i) collecting a plasma sample from an HIV-infected patient;(ii) amplifying the HIV-encoding RNA in the plasma sample by convertingthe XHA to cDNA and amplifying HIV sequences using HIV primers thatresult in a PCR product that comprises the RT gene; (iii) performing PCRusing primers that result in PCR products comprising the wild type ormutations at codon 98 and 181; and (iv) determining, via the products ofPCR, the presence or absence of a mutation at codon 98 and 181.

Yet another preferred, non-limiting specific embodiment of the inventionis as follows: A method of assessing the effectiveness of NNRTI therapyof a patient comprising (i) collecting a plasma sample from anHIV-infected patient; (ii) amplifying the HIV-encoding RNA in the plasmasample by converting the SKA. to cDNA and amplifying HIV sequences usingHIV primers that result in a PCR product that comprises the RT gene;(iii) performing PCR using primers that result in PCR productscomprising the wild type or mutations at codon 101 and 190; and (iv)determining, via the products of PCR, the presence or absence of amutation at codon 190, for example 190S and 101.

Yet another preferred, non-limiting specific embodiment of the inventionis as follows: A method of assessing the effectiveness of NNRTI therapyof a patient comprising (i) collecting a plasma sample from anHIV-infected patient; (ii) amplifying the HIV-encoding RNA in the plasmasample by converting the RNA to cDNA and amplifying HIV sequences usingHIV primers that result in a PCR product that comprises the RT gene;(iii) performing PCR using primers that result in PCR productscomprising the wild type or a mutation at codon 108; and (iv)determining, via the products of PCR, the presence or absence of amutation at codon 108. Yet another preferred, non-limiting specificembodiment of the invention is as follows: A method of assessing theeffectiveness of NNRTI therapy of a patient comprising (i) collecting aplasma sample from an HIV-infected patient; (ii) amplifying theHIV-encoding RNA in the plasma sample by converting the RNA to cDNA andamplifying HIV sequences using HIV primers that result in a PCR productthat comprises the RT gene; (iii) performing PCR using primers thatresult in PCR products comprising the wild type or mutations at codon101 and 103 and 190 and (iv) determining, via the products of PCR, thepresence or absence of a mutation at codon 101 and 103 and 190, forexample 190A. Yet another preferred, non-limiting specific embodiment ofthe invention is as follows: A method of assessing the effectiveness ofNNRTI therapy of a patient comprising (i) collecting a plasma samplefrom an HIV-infected patient; (ii) amplifying the HIV-encoding RNA inthe plasma sample by converting the RNA to cDNA and amplifying HIVsequences using HIV primers that result in a PCR product that comprisesthe RT gene; (iii) performing PCR using primers that result in PCRproducts comprising the wild type or mutations at codon 106 and 189 and181 and 227 and (iv) determining, via the products of PCR, the presenceor absence of a mutation at codon 106 and 189 and 181 and 227. Yetanother preferred, non-limiting specific embodiment of the invention isas follows: A method of assessing the effectiveness of NNRTI therapy ofa patient comprising (i) collecting a plasma sample from an HIV-infectedpatient; (ii) amplifying the HIV-encoding RNA in the plasma sample byconverting the RNA to cDNA and amplifying HIV sequences using HIVprimers that result in a PCR product that comprises the RT gene; (iii)performing PCR using primers that result in PCR products comprising thewild type or mutations at codon 188 and 100 and 103 and (iv)determining, via the products of PCR, the presence or absence of amutation at codon 188 and 100 and 103.

The presence of the mutation at codon 225 and 103 of HIV RT indicatesthat the effectiveness of the current or prospective NNRTI therapy mayrequire alteration, since as shown by this invention mutation at codon103 reduces susceptibility which susceptibility can in part be restoredby mutation at codon 225. Using the methods of this invention change inthe NNRTI therapy would be indicated. Similarly, using the means andmethods of this invention the presence of the mutation at codon 236 and103 and/or 181 of the HIV RT indicates that the effectiveness of thecurrent or prospective NNRTI therapy has been diminished. Similarly,using the means and methods of this invention the presence of themutation at codon 190 (G190A) and 103 (K103N) of the HIV RT indicatesthat the effectiveness of the current or prospective NNRTI therapy hasbeen diminished. Similarly, using the means and methods of thisinvention the presence of the mutation at codon 190 (G190S) and 101(K101E) of the HIV RT indicates that the effectiveness of the current orprospective NNRTI therapy baa been diminished. Similarly, using themeans and methods of this invention the presence of the mutation atcodon 230 and 181 of the HIV RT indicates that the effectiveness of thecurrent or prospective NNRTI therapy has been diminished. Similarly,using the means and methods of this invention the presence of the amutation at codon 181 of the HIV RT indicates that the effectiveness ofthe current or prospective NNRTI therapy has been diminished. Similarly,using the means and methods of this invention the presence of themutation at codon 188 of the HIV RT indicates that the effectiveness ofthe current or prospective NNRTI therapy has been diminished. Similarly,using the means and methods of this invention the presence of themutation at codon 138 and 188 of the HIV RT indicates that theeffectiveness of the current or prospective NNRTI therapy has beendiminished. Similarly, using the means and methods of this invention thepresence of the mutation at codon 98 of the HIV RT indicates that theeffectiveness of the current or prospective NNRTI therapy has beendiminished. Similarly, using the means and methods of this invention thepresence of the mutation at codon 98 and 190 of the HIV RT indicatesthat the effectiveness of the current or prospective NNRTI therapy hasbeen diminished. Similarly, using the means and methods of thisinvention the presence of the mutation at codon 181 and 98 of the HIV RTindicates that the effectiveness of the current or prospective NNRTItherapy has been diminished. Similarly, using the means and methods ofthis invention the presence of the mutation at codon 101 and 190, forexample 190S, of the HIV RT indicates that the effectiveness of thecurrent or prospective NNRTI therapy has been diminished. Similarly,using the means and methods of this invention the presence of a mutationat codon 108 of the HIV RT indicates that the effectiveness of thecurrent or prospective NNRTI therapy has been diminished. Similarly,using the means and methods of this invention the presence of themutations at 101 and 103 and 190, for example 190A, of the HIV RTindicates that the effectiveness of the current or prospective NNRTItherapy has been diminished. Similarly, using the means and methods ofthis invention the presence of the mutation at codon 106 and 189 and 181and 227 of the HIV RT indicates that the effectiveness of the current orprospective NNRTI therapy has been diminished. Similarly, using themeans and methods of this invention the presence of the mutation atcodon 188 and 100 and 103 of the HIV RT indicates that the effectivenessof the current or prospective NNRTI therapy has been diminished.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of evaluating the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 236 and 103 and/or 181.Using the phenotypic susceptibility assay, it was observed that thepresence of the three mutations correlates positively with delavirdineresistance. Using the phenotypic susceptibility assay, it was observedthat the presence of the three mutations correlates positively withnevirapine resistance. In another embodiment, the mutated codon 236 ofHIV RT encodes leucine (L). In a further embodiment, the reversetranscriptase has a mutation at codon 103, a mutation at codon 181 or acombination thereof in addition to the mutation at codon 236 of HIV RT.In a still further embodiment, the mutated codon 103 encodes anasparagine (N) and the mutated codon at 181 encodes a cysteine (C).

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 225 and 103. Using thephenotypic susceptibility assay, it was observed that the presence ofthe mutations at codons 225 alone or in combination with a mutation atcodon 103 of HIV RT cause an increase in delavirdine susceptibility,while having no effect on nevirapine susceptibility. In yet anotherembodiment, the mutated codon 225 codes for a histidine.

This invention provides a method of assessing the effectiveness ofantiretroviral therapy of an HIV-infected patient comprising: (a)collecting a biological sample from an HIV-infected patient; and (b)determining whether the biological, sample comprises nucleic acidencoding HIV reverse transcriptase having a mutation at codon 181. Usingthe phenotypic susceptibility assay it was observed that the presence ofmutations at codon 181 correlates positively with a moderate decrease indelavirdine susceptibility and a significant decrease in nevirapinesusceptibility and no change in efavirenz susceptibility. In anembodiment, the mutated colon 181 codes for a isoleuecine.

This invention provides a method of assessing the effectiveness ofantiretroviral therapy of an HIV-infected patient comprising: (a)collecting a biological sample from an HIV-infected patient; and (b)determining whether the biological sample comprises nucleic acidencoding HIV reverse transcriptase having a mutation at codon 188. Usingthe phenotypic susceptibility assay it was observed that the presence ofmutations at colon 188 correlates positively with a slight decrease indelavirdine susceptibility and a substantial decrease in nevirapinesusceptibility and significant decrease in efavirenz susceptibility. Inan embodiment, the mutated codon 188 codes for a cysteine, histidine, orleucine.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 138 and 188. Using thephenotypic susceptibility assay, it was observed that the presence ofthe mutations at codons 138 alone or in combination with a mutation atcodon 188 of HIV RT causes a moderate decrease in delavirdinesusceptibility and a substantial decrease in nevirapine susceptibilityand a moderate decrease in efavirenz susceptibility. In yet anotherembodiment, the mutated codon 138 codes for a alanine and codon 188codes for a leucine.

This invention provides a method of assessing the effectiveness ofantiretroviral therapy of an HIV-infected patient comprising: (a)collecting a biological sample from an HIV-infected patient; and (b)determining whether the biological sample comprises nucleic acidencoding HIV reverse transcriptase having a mutation at codon 98. Usingthe phenotypic susceptibility assay it was observed that the presence ofmutations at codon 98 correlates positively with a slight decrease indelavirdine susceptibility and a slight decrease in nevirapinesusceptibility and a slight decrease in efavirenz susceptibility. In anembodiment, the mutated codon 98 codes for a glycine.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 98 and 190. Using thephenotypic susceptibility assay, it was observed that the presence ofthe mutations at codons 98 alone or in combination with a mutation atcodon 190 of HIV RT causes an increase in delavirdine susceptibility anda substantial decrease in nevirapine susceptibility and a substantialdecrease in efavirenz susceptibility. In yet another embodiment, themutated codon 190 codes for a serine and codon 98 codes for a glycine.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 181 and 98. Using thephenotypic susceptibility assay, it was observed that the presence ofthe mutations at codons 181 alone or in combination with a mutation atcodon 98 of HIV RT causes a significant decrease in delavirdinesusceptibility and a substantial decrease in nevirapine susceptibilityand a slight decrease in efavirenz susceptibility. In yet anotherembodiment, the mutated codon 98 codes for a glycine and codon 181 codesfor a cysteine.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 101 and 190, forexample 190S. Using the phenotypic susceptibility assay, it was observedthat the presence of the mutations at codons 101 alone or in combinationwith a mutation at codon 190 of HIV RT causes no change in delavirdinesusceptibility and a substantial decrease in nevirapine susceptibilityand a substantial decrease in efavirenz susceptibility. In yet anotherembodiment, the mutated codon 190 codes for a serine and codon 101 codesfor a glutamine acid.

This invention provides a method of assessing the effectiveness ofantiretroviral therapy of an HIV-infected patient comprising: (a)collecting a biological sample from an HIV-infected patient; and (b)determining whether the biological sample comprises nucleic acidencoding HIV reverse transcriptase having a mutation at codon 108. Usingthe phenotypic susceptibility assay It was observed that the presence ofmutations at codon 108 correlates positively with no change indelavirdine susceptibility and a slight decrease in nevirapinesusceptibility and no change in efavirenz susceptibility. in anembodiment, the mutated codon 108 codes for a isoleucine.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 101 and 190, forexample 190A. Using the phenotypic susceptibility assay, it was observedthat the presence of the mutations at codons 101 alone or in combinationwith a mutation at codon 190 of HIV RT causes no change in delavirdinesusceptibility and a substantial decrease in nevirapine susceptibilityand a significant decrease in efavirenz susceptibility. In yet anotherembodiment, the mutated codon 190 codes for a glycine and codon 101codes for a glutamine acid.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 103 and 190. Using thephenotypic susceptibility assay, it was observed that the presence ofthe mutations at codons 103 alone or in combination with a mutation atcodon 190 of HIV RT causes a moderate decrease in delavirdinesusceptibility and a substantial decrease in nevirapine susceptibilityand a significant decrease in efavirenz susceptibility. In yet anotherembodiment, the mutated codon 190 codes for a alanine and codon 103codes for a asparagine. Another preferred, non-limiting, specificembodiment of the invention is as follows: a method of assessing theeffectiveness of antiretroviral therapy of an HIV-infected patientcomprising: (a) collecting a biological sample from an HIV-infectedpatient; and (b) determining whether the biological sample comprisesnucleic acid encoding HIV reverse transcriptase having a mutation atcodon 106 and 181. Using the phonotypic susceptibility assay, it wasobserved that the presence of the mutations at codons 106 alone or incombination with a mutation at codon 181 of HIV RT causes a significantdecrease in delavirdine susceptibility and a substantial decrease innevirapine susceptibility and a slight decrease in efavirenzsusceptibility. In yet another embodiment, the mutated codon 106 codesfor a alanine and codon 181 codes for a cysteine.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 106 and 189. Using thephenotypic susceptibility assay, it was observed that the presence ofthe mutations at codons 106 alone or in combination with a mutation atcodon 189 of HIV RT causes a slight decrease in delavirdinesusceptibility and a moderate decrease in nevirapine susceptibility andno change in efavirenz susceptibility. In yet another embodiment, themutated codon 189 codes for a leucine and a codon 106 codes for aalanine.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 106 and 227. Using thephenotypic susceptibility assay, it was observed that the presence ofthe mutations at codons 106 alone or in combination with a mutation atcodon 227 of HIV RT causes a slight decrease in delavirdinesusceptibility and a substantial decrease in nevirapine susceptibilityand a slight decrease in efavirenz susceptibility. In yet anotherembodiment, the mutated codon 227 codes for a leucine and codon 106codes for a alanine.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 181 and 227. Using thephenotypic susceptibility assay, it was observed that the presence ofthe mutations at codon 181 alone or in combination with a mutation atcodon 227 of HIV RT causes an increase in delavirdine susceptibility anda significant decrease in nevirapine susceptibility and an increase inefavirenz susceptibility. In yet another embodiment, the mutated codon227 codes for a leucine and codon 181 codes for a cysteine.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 106 and 181 and 227.Using the phenotypic susceptibility assay, it was observed that thepresence of the mutations at codons 106 alone or in combination with amutation at codon 181 and 227 of HIV RT causes a moderate decrease indelavirdine susceptibility and a substantial decrease in nevirapinesusceptibility and a slight decrease in efavirenz susceptibility. In yetanother embodiment, the mutated codon 106 codes for a alanine, codon 181codes for a cysteine and codon 227 codes for a leucine.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 103 and 188. Using thephenotypic susceptibility assay, it was observed that the presence ofthe mutations at codons 103 alone or in combination with a mutation atcodon 188 of HIV RT causes a substantial decrease in delavirdinesusceptibility and a substantial decrease in nevirapine susceptibilityand a substantial decrease in efavirenz susceptibility. In yet anotherembodiment, the mutated codon 188 codes for a leucine and codon 103codes for a asparagine.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 100 and 103. Using thephenotypic susceptibility assay, it was observed that the presence ofthe mutations at codons 100 alone or in combination with a mutation atcodon 103 of HIV RT causes a substantial decrease in delavirdinesusceptibility and a moderate decrease in nevirapine susceptibility anda substantial decrease in efavirenz susceptibility. In yet anotherembodiment, the mutated codon 100 codes for a isoleucine and codon 103codes for a asparagine.

Another preferred, non-limiting, specific embodiment of the invention isas follows: a method of assessing the effectiveness of antiretroviraltherapy of an HIV-infected patient comprising: (a) collecting abiological sample from an HIV-infected patient; and (b) determiningwhether the biological sample comprises nucleic acid encoding HIVreverse transcriptase having a mutation at codon 100 and 103 and 188.Using the phenotypic susceptibility assay, it was observed that thepresence of the mutations at codons 100 alone or in combination with amutation at codon 103 and 188 of HIV RT causes a substantial decrease indelavirdine susceptibility and a substantial decrease in nevirapinesusceptibility and a substantial decrease in efavirenz susceptibility.In yet another embodiment, the mutated codon 100 codes for a isoleucine,codon 103 codes for a asparagine and codon 188 codes for a leucine.

This invention also provides the means and methods to use the resistancetest vector comprising an HIV gene further comprising an NNRTI mutationfor drug screening. More particularly, the invention describes theresistance test vector comprising the HIV reverse transcriptase havingmutations at codons 225 and 103 for drug screening. The invention alsodescribes the resistance test vector comprising the HIV reversetranscriptase having mutations at codons 236 and 103 and/or 181. Theinvention also describes the resistance test vector comprising the HIVreverse transcriptase having mutations at codons 190 (G190A) and 103(K103N). The invention also describes the resistance test vectorcomprising the HIV reverse transcriptase having mutations at codons 190(G190S) and 101 (K101E). The invention also describes the resistancetest vector comprising the HIV reverse transcriptase having mutations atcodons 230 and 181. The invention also describes the resistance testvector comprising the HIV reverse transcriptase having a mutation atcodon 181. The invention also describes the resistance test vectorcomprising the HIV reverse transcriptase having a mutation at codon 188.The invention also describes the resistance test vector comprising theHIV reverse transcriptase having mutations at codons 138 and 188. Theinvention also describes the resistance test vector comprising the HIVreverse transcriptase having a mutation at 98. The invention alsodescribes the resistance test vector comprising the HIV reversetranscriptase having mutations at codons 98 and 190. The invention alsodescribes the resistance test vector comprising the HIV reversetranscriptase having mutations at codons 181 and 98. The invention alsodescribes the resistance test vector comprising the HIV reversetranscriptase having mutations at codons 101 and 190, for example 190S.The invention also described the resistance test vector comprising theHIV reverse transcriptase having a mutation at codon 108. The inventionalso describes the resistance teat vector comprising the HIV reversetranscriptase having mutations at codons 101 and 103 and/or 190, forexample 190A. The invention also describes the resistance test vectorcomprising the HIV reverse transcriptase having mutations at codons 106and 189 and/or 181 and/or 227. The invention also describes theresistance test vector comprising the HIV reverse transcriptase havingmutations at codons 188 and 100 and/or 103. The invention furtherrelates to novel vectors, host cells and compositions for isolation andidentification of the non-nucleoside HIV-1 reverse transcriptaseinhibitor resistance mutant and using such vectors, host cells andcompositions to carry out anti-viral drug screening. This invention alsorelates to the screening of candidate drugs for their capacity toinhibit said mutant.

EXAMPLE 1 Phenotypic Drug Susceptibility and Resistance Test UsingResistance Test Vectors

Phenotypic drug susceptibility and resistance tests are carried outusing the means and methods described in PCT International ApplicationNo. PCT/US97/01609, filed Jan. 29, 1997 which is hereby incorporated byreference.

In these experiments patient-derived segment(s) corresponding to the HIVprotease and reverse transcriptase coding regions were eitherpatient-derived segments amplified by the reversetranscription-polymerase chain reaction method (RT-PCR) using viral RNAisolated from viral particles present in the serum of HIV-infectedindividuals or were mutants of wild type HIV-1 made by site directedmutagenesis of a parental clone of resistance test vector DNA. Isolationof viral RNA was performed using standard procedures (e.g. RNAgentsTotal RNA Isolation System, Promega, Madison Wis. or RNAzol, Tel-Test,Friendswood, Tex.). The RT-PCR protocol was divided into two steps. Aretroviral reverse transcriptase [e.g. Moloney MuLV reversetranscriptase (Roche Molecular Systems, Inc., Branchburg, N.J.), oravian myeloblastosis virus (AMV) reverse transcriptase, (BoehringerMannheim, Indianapolis, Ind.)] was used to copy viral RNA into cDNA. ThecDNA was then amplified using a thermostable DNA polymerase [e.g. Taq(Roche Molecular Systems, Inc., Branchburg, N.J.), Tth (Roche MolecularSystems, Inc., Branchburg, N.J.), PrimeZyme (isolated from Thermusbrockianus, Biometra, Gottingen, Germany)] or a combination ofthermostable polymerases as described for the performance of “long PCR”(Barnes, W. M., (1994) Proc. Natl. Acad. Sci, USA 91, 2216-2220) [e.g.Expand High Fidelity PCR System (Taq+Pwo), (Boehringer Mannheim,Indianapolis, Ind.) OR GeneAmp XL PCR kit (Tth+Vent), (Roche MolecularSystems, Inc., Branchburg, N.J.)].

The primers, ApaI primer (PDSApa) and AgeI primer (PDSAge) used toamplify the “test” patient-derived segments contained sequencesresulting in ApaI and AgeI recognition sites being introduced into the5′ and 3′ termini of the PCR product, respectively as described in PCTInternational Application No. FCT/US97/01609, filed Jan. 29, 1997.

Resistance test vectors incorporating the “test” patient-derivedsegments were constructed as described in PCT International ApplicationNo. PCT/US97/01609, filed Jan. 29, 1997 using an amplified DNA productof 1.5 kB prepared by RT-PCR using viral RNA as a template andoligonucleotides PDSApa (1) and PDSAge (2) as primers, followed bydigestion with ApaI and AgeI or the isoschizimer PINAI. To ensure thatthe plasmid DNA corresponding to the resultant resistance test vectorcomprises a representative sample of the HIV viral quasi-species presentin the serum of a given patient, many (>100) independent E. colitransformants obtained in the construction of a given resistance testvector were pooled and used for the preparation of plasmid DNA.

A packaging expression vector encoding an amphotrophic MuLV 4070A envgene product enables production in a resistance test vector host cell ofresistance test vector viral particles which can efficiently infecthuman target cells. Resistance test vectors encoding all HIV genes withthe exception of env were used to transfect a packaging host cell (oncetransfected the host cell is referred to as a resistance test vectorhost cell). The packaging expression vector which encodes theamphotrophic MuLV 4070A env gene product is used with the resistancetest vector to enable production in the resistance test vector host cellof infectious pseudotyped resistance test vector viral particles.

Resistance tests performed with resistance test vectors were carried outusing packaging host and target host cells consisting of the humanembryonic kidney cell line 293 (Cell Culture Facility, UC San Francisco,SF, Calif.) or the Jurkat leukemic T-cell line (Arthur Weiss, UC SanFrancisco, SF, Calif.).

Resistance tests were carried out with resistance test vectors using twohost cell types. Resistance test vector viral particles were produced bya first host cell (the resistance test vector host cell) that wasprepared by transfecting a packaging host cell with the resistance testvector and the packaging expression vector. The resistance test vectorviral particles were then used to infect a second host cell (the targethost cell) in which the expression of the indicator gene is measured.

The resistance test vectors containing a functional luciferase genecassette were constructed and host cells were transfected with theresistance test vector DNA. The resistant test vectors containedpatient-derived reverse transcriptase and protease sequences that wereeither susceptible or resistant to the antiretroviral agents, such asnucleoside reverse transcriptase inhibitors, non-nucleoside reversetranscriptase inhibitors and protease inhibitors. The resistance testvector viral particles produced by transfecting the resistance testvector DNA into host cells, either in the presence or absence ofprotease inhibitors, were used to infect target host cells grown eitherin the absence of NRTI or NNRTI or in the presence of increasingconcentrations of the drug. The amount of luciferase activity producedin infected target host cells in the presence of drug was compared tothe amount of luciferase produced in infected target host cells in theabsence of drug. Drug resistance was measured as the amount of drugrequired to inhibit by 50% the luciferase activity detected in theabsence of drug (inhibitory concentration 50%, IC50). The IC50 valueswere determined by plotting percent drug inhibition vs. log drugconcentration.

Host cells were seeded in 10-cm-diameter dishes and were transfectedseveral days after plating with resistance test vector plasmid DNA andthe envelope expression vector. Transfections were performed using acalcium-phosphate precipitation procedure. The cell culture mediacontaining the DNA precipitate was replaced with fresh medium, from oneto 24 hours, after transfection. Cell culture media containingresistance test vector viral particles was harvested one to four daysafter transfection and was passed through a 0.45-mm filter before beingstored at 80° C. HIV capsid protein (p24) levels in the harvested cellculture media were determined by an EIA method as described by themanufacturer (SIAC; Frederick, Md.). Before infection, target cells (293and 293/T) were plated in cell culture media. Control infections wereperformed using cell culture media from mock transfections (no DNA) ortransfections containing the resistance test vector plasmid DNA withoutthe envelope expression plasmid. One to three or more days afterinfection the media was removed and cell lysis buffer (Promega) wasadded to each well. Cell lysates were assayed for luciferase activity(FIG. 3). The inhibitory effect of the drug was determined using thefollowing equation:luciferase inhibition=1−(RLUluc[drug]÷RLUluc)×100where RLUluc [drug] is the relative light unit of 25 luciferase activityin infected cells in the presence of drug and RLUluc is the RelativeLight Unit of luciferase activity in infected cells in the absence ofdrug. IC50 values were obtained from the sigmoidal curves that weregenerated from the data by plotting the percent inhibition of luciferaseactivity vs. the log drug concentration. The drug inhibition curves areshown in (FIG. 3).

EXAMPLE 2 Correlating Phenotypic Susceptibility and Genotypic Analysis

Phenotypic Susceptibility Analysis of Patient HIV Samples

Resistance test vectors are constructed as described in example 1.Resistance test vectors, or clones derived from the resistance testvector pools, are tested in a phenotypic assay to determine accuratelyand quantitatively the level of susceptibility to a panel ofanti-retroviral drugs. This panel of anti-retroviral drugs may comprisemembers of the classes known as nucleoside-analog reverse transcriptaseinhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors(NNRTIs), and protease inhibitors (PRIs). The panel of drugs can beexpanded as new drugs or new drug targets become available. An IC50 isdetermined for each resistance test vector pool for each drug tested.The pattern of susceptibility to all of the drugs tested is examined andcompared to known patterns of susceptibility. A patient sample can oefurther examined for genotypic changes correlated with the pattern ofsusceptibility observed.

Genotypic Analysis of Patient HIV Samples

Resistance test vector DNAs, either pools or clones, are analyzed by anyof the genotyping methods described in Example 2. In one embodiment ofthe invention, patient HIV sample sequences are determined using viralRNA purification, RT/PCR and ABI chain terminator automated sequencing.The sequence that is determined is compared to control sequences presentin the database or is compared to a sample from the patient prior toinitiation of therapy, if available. The genotype is examined forsequences that are different from the control or pre-treatment sequenceand correlated to the observed phenotype.

Phenotypic Susceptibility Analysis of Site Directed Mutants

Genotypic changes that are observed to correlate with changes inphenotypic patterns of drug susceptibility are evaluated by constructionof resistance test vectors containing the specific mutation on adefined, wild-type 35 (drug susceptible) genetic background. Mutationsmay be incorporated alone and/or in combination with other known drugresistance mutations that are thought to modulate the susceptibility ofHIV to a certain drug or class of drugs. Mutations are introduced intothe resistance test vector through any of the widely known methods forsite-directed mutagenesis. In one embodiment of this invention themega-primer PCR method for site-directed mutagenesis is used. Aresistance test vector containing the specific mutation or group ofmutations is then tested using the phenotypic susceptibility assaydescribed above and the susceptibility profile is compared to that of agenetically defined wild-type (drug susceptible) resistance test vectorwhich lacks the specific mutations. Observed changes in the pattern ofphenotypic susceptibility to the antiretroviral drugs tested isattributed to the specific mutations introduced into the resistance testvector.

EXAMPLE 3 Correlating Phenotypic Susceptibility and Genotypic Analysis:P225H

Phenotypic Analysis of Patient 97-302

A resistance test vector was constructed as described in example 1 froma patient sample designated as 97-302. This patient had been treatedwith d4T, indinavir and DMP-266 for a period of approximately 10 months.Isolation of viral RNA and RT/PCR was used to generate a patient derivedsegment that comprised viral sequences coding for all of PR and aa 1-313of RT. The patient derived segment was inserted into a indicator geneviral vector to generate a resistance test vector designated RTV-302.RTV-302 was tested using a phenotypic susceptibility assay to determineaccurately and quantitatively the level of susceptibility to a panel ofanti-retroviral drugs. This panel of anti-retroviral drugs comprisedmembers of the classes known as NRTIs (AZT, 3TC, d4T, ddI and ddC),NNRTIs (delavirdine and nevirapine), and PRIs (indinavir, nelfinavir,ritonavir, and saquinavir). An IC50 was determined for each drug tested.Susceptibility of the patient virus to each drug was examined andcompared to known patterns of susceptibility. A pattern ofsusceptibility to the NNRTIs was observed for patient sample RTV-302 inwhich there was significant decrease in nevirapine susceptibility(increased resistance) and modest decrease in delavirdine susceptibility(See FIG. 8A). Patient sample 97-302 was examined further for genotypicchanges associated with the observed pattern of susceptibility.

Determination of Genotype of Patient 97-302

RTV-302 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database, Los Alamos, N. Mex.). Thenucleotide sequence was examined for sequences that are different fromthe control sequence. RT mutations were noted at positions K103N, I135M,T200A, am P225H. K103N is associated with resistance to the NNRTIs andhas been shown using the phenotypic susceptibility assay to beassociated with reduced susceptibility to both delavirdine andnevirapine to an equal extent. The mutations at I135M and T200A areknown polymorphisms of the wild-type (drug-sensitive) variants of HIV.The mutation, P225H was characterized using site directed mutagenesisand phenotypic susceptibility testing to correlate the changes at aminoacid 225 with changes In NNRTI phenotypic susceptibility.

Site Directed Mutagenesis

Resistance test vectors were constructed containing the P225H mutationalone and in combination with other known drug resistance mutations(K103N, Y181C) known to modulate the HIV susceptibility to NNRTIs.Mutations were introduced into the resistance test vector using themega-primer PCR method for site-directed mutagenesis. (Sakar G andSommar S S (1994) Biotechniques 8(4), 404-407). A resistance test vectorcontaining the P225H mutation (P225H-RTV) was tested using thephenotypic susceptibility assay described above and the results werecompared to that of a genetically defined resistance test vector thatwas wild type at position 225. The pattern of phenotypic susceptibilityto the NNRTI, delavirdine in the P225H-RTV was altered as compared towild type. In the context of an otherwise wild type background (i.e.P225H mutation alone) the P225H-RTV was more susceptible to delavirdinethan the wild type control RTV. No significant change in nevirapinesusceptibility was observed in the P225H-RTV. The P225H mutation wasalso introduced into a RTV containing additional mutations at K103N,Y181C or both (K103N+Y181C). In all cases, RTVs were more susceptible toinhibition by delavirdine if the P225H mutation was present as comparedto the corresponding RTV lacking the P225H mutation (FIG. 8D). In allcases the P225H mutation did not significantly change nevirapinesusceptibility (FIG. 8D).

EXAMPLE 4 Correlating Phenotypic Susceptibility and Genotypic Analysis:P236L

Phenotypic Analysis of HIV Patient 97-268

A resistance test vector was constructed as described in Example 11 froma patient sample designated 97-268. This patient had been treated withAZT and 3TC (NRTIs), indinavir and saquinavir (PRIs) and delavirdine (anNNRTI) for periods varying from 1 month to 2 years. Isolation of viralRNA and RT/PCR was used to generate a patient derived segment thatcomprised viral sequences coding for all of PR and amino acids 1-313 ofRT. The patient derived segment was inserted into a indicator gene viralvector to generate a resistance test vector designated RTV-268. RTV-268was then tested using the phenotypic susceptibility assay to determineaccurately and quantitatively the level of susceptibility to a panel ofanti-retroviral drugs. This panel of anti-retroviral drugs comprisedmembers of the classes known as NRTIs (AZT, 3TC, d4T, ddI and ddC),NNRTIs (delavirdine and nevirapine), and PRIs (indinavir, nelfinavir,ritonavir, and saquinavir). An IC50 was determined for each drug tested.Susceptibility of the patient virus to each drug was examined andcompared to the susceptibility of a reference virus. A pattern ofsusceptibility to the NNRTIs was observed for the patient sample RTV-268in which the virus sample was observed to be resistant to delavirdinewith no resistance to delavirdine. The sample was examined further forgenotypic changes associated with the pattern of susceptibility.

Genotype of HIV Patient 97-268

RTV-268 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of wildtype clade B HIV-1. The nucleotide sequence was evaluated for sequencesdifferent from the control sequence. RT mutations were noted atpositions M41L, D67N, M184V, T200A, E203D, L210W, T215Y, K219Q, andP236L compared to the control sequence. The mutations at T200A and E203Dare known polymorphisms in wild-type (drug-sensitive) variants of HIV.Mutations at positions M41L, D67N, L210W, T215Y, and K219Q areassociated with AZT resistance. The mutation at M184V is associated with3TC resistance. The mutation at P236L is associated with resistance todelavirdine and increased susceptibility to nevirapine (Dueweke et al.,Ibid.). In contrast to previous reports, the RTV-268 sample showed nochange in nevirapine susceptibility. The mutation, P236L, wascharacterized using site directed mutagenesis and in vitro phenotypicsusceptibility testing to correlate changes at amino acid 236 withchanges in phenotypic susceptibility.

Site Directed Mutagenesis

Resistance test vectors were constructed containing the P236L mutationalone and in combination with other known drug resistance mutations(K103N, Y181C) that are known to modulate the susceptibility of HIV-1 toNNRTIs. Mutations were introduced into the resistance test vector usingthe mega-primer PCR. method for site-directed mutagenesis (Sakar andSommar, Ibid.). A resistance test vector containing the P236L mutation(P236L-RTV) was tested using the phenotypic susceptibility assay and theresults were compared to that of a genetically defined resistance testvector that was wild type at position 236. P236L-RTV exhibited changesin NNRTI phenotypic susceptibility. In the context of an otherwise wildtype background (i.e. P236L mutation alone) the P236L-RTV is lesssusceptible to delavirdine than a wild type reference RTV. In contrastto Dueweke et al. no significant change in nevirapine susceptibility wasobserved for P236L-RTV. The P236L mutation was also introduced into aRTV containing mutations at K103N, Y181C or both (K103N+Y181C). In allcases, the RTVs were less susceptible (more resistant) to delavirdine ifthe P236L mutation was present as compared to the corresponding RTVlacking the P236L mutation. In all cases the P236L mutation did notsignificantly alter nevirapine susceptibility.

EXAMPLE 5 Correlating Phenotypic Susceptibility and Genotypic Analysis:G190S

Phenotypic Analysis of HIV Patient 97-644

A resistance test vector was constructed as described in Example 1 froma patient sample designated 97-644. This patient had been treated withd4T (NRTI), indinavir (PRI) and efavirenz (NNRTI). for a period varyingfrom to 17 months. Isolation of viral RNA and RT/PCR was used togenerate a patient derived segment that comprised viral sequences codingfor all of PR and amino acids 1-313 of RT. The patient derived segmentwas inserted into a indicator gene viral vector to generate a resistancetest vector designated RTV-644. RTV-644 was then tested using thephenotypic susceptibility assay to determine accurately andquantitatively the level of susceptibility to a panel of anti-retroviraldrugs. This panel of anti-retroviral drugs comprised members of theclasses known as NRTIs (AZT, 3TC, d4T, ddI and ddC), NNRTIs (delavirdineand nevirapine), and PRIs (indinavir, nelfinavir, ritonavir, andsaquinavir). An IC50 was determined for each drug tested. Susceptibilityof the patient virus to each drug was examined and compared to thesusceptibility of a reference virus. A pattern of susceptibility to theNNRTIs was observed for the patient sample RTV-644 in which the virussample was observed to be resistant to nevirapine with little or noresistance to delavirdine. The sample was examined further for genotypicchanges associated with the pattern of susceptibility.

Genotype of HIV Patient 97-644

RTV-644 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of wildtype clade B HIV-1. The nucleotide sequence was evaluated for sequencesdifferent from the control sequence. RT mutations were noted atpositions K101E and G190S compared to the control sequence. Themutations at T200A and E203D are known polymorphisms in wild-type(drug-sensitive) variants of HIV. The mutation at K101E is associatedwith resistance to some but not all NNRTIs. The mutation G190A but notspecifically G190S is associated with nevirapine and lovirideresistance. The mutations G190S and G190A were characterized using sitedirected mutagenesis and in vitro phenotypic susceptibility testing tocorrelate changes at amino acid 190 with changes in phenotypicsusceptibility.

Site Directed Mutagenesis

Resistance test vectors were constructed containing the G190S and G190Amutations. Mutations were introduced into the resistance test vectorusing the mega-primer PCR method for site-directed mutagenesis (Sakarand Sommar, Ibid.). Resistance test vectors containing the G190S orG190A mutations (G190S-RTV, or G190A-RTV) were tested using thephenotypic susceptibility assay and the results were compared to that ofa genetically defined resistance test vector that was wild type atposition G190. G190S-RTV and G190A-RTV exhibited changes in NNRTIphenotypic susceptibility. In the context of an otherwise wild typebackground these RTVs were markedly less susceptible to nevirapine andslightly more susceptible to delavirdine than a wild type referenceRTTr.

EXAMPLE 6 Predicting Response to Non-Nucleoside Reverse TranscriptaseInhibitors by Characterization of Amino Acid Changes in HIV-1 ReverseTranscriptase

Phenotypic and Genotypic Correlation of Mutations at Amino Acid 236 ofHIV-1 Reverse Transcriptase

In one embodiment of this invention, changes in the amino acid atposition 236 of the reverse transcriptase protein of HIV-1 is evaluatedusing the following method comprising: (i) collecting a biologicalsample from an HIV-1 infected subject; (ii) evaluating whether thebiological sample contains nucleic acid encoding HIV-1 reversetranscriptase having a mutation at codon 236. The presence of a mutationat codon 236 (P236L) is correlated with a reduction in delavirdinesusceptibility and little or no change in nevirapine susceptibility.

The biological sample comprises whole blood, blood components includingperipheral mononuclear cells (PBMC), serum, plasma (prepared usingvarious anticoagulants such as EDTA, acid citrate-dextrose, heparin),tissue biopsies, cerebral spinal fluid (CSF), or other cell, tissue orbody fluids. In another embodiment, the HIV-1 nucleic acid (genomic RNA)or reverse transcriptase protein can be isolated directly from thebiological sample or after purification of virus particles from thebiological sample. Evaluating whether the amino acid at position 236 ofthe HIV-1 reverse transcriptase is mutated, can be performed usingvarious methods, such as direct characterization of the viral nucleicacid encoding reverse transcriptase or direct characterization of thereverse transcriptase protein itself. Defining the amino acid atposition 236 of reverse transcriptase can be performed by directcharacterization of the reverse transcriptase protein by conventional ornovel amino acid sequencing methodologies, epitope recognition byantibodies or other specific binding proteins or compounds.Alternatively, the amino acid at position 236 of the HIV-1 reversetranscriptase protein can be defined by characterizing amplified copiesof HIV-1 nucleic acid encoding the reverse transcriptase protein.Amplification of the HIV-1 nucleic acid can be performed using a varietyof methodologies including reverse transcription-polymerase chainreaction (RT-PCR), NASBA, SDA, RCR, or 3SR as would be known to theordinarily skilled artisan. Evaluating whether the nucleic acid encodingHIV reverse transcriptase has a mutation at codon 236 can be performedby direct nucleic acid sequencing using various primer extension-chaintermination (Sanger, ABI/PE and Visible Genetics) or chain cleavage(Maxam and Gilbert) methodologies or more recently developed sequencingmethods such as matrix assisted laser desorption-ionization time offlight (MALDI-TOF) or mass spectrometry (Sequenom, Gene Trace Systems).Alternatively, the nucleic acid sequence encoding amino acid position236 can be evaluated using a variety of probe hybridizationmethodologies, such as genechip hybridization sequencing (Affymetrix),line probe assay (LiPA; Murex), and differential hybridization (Chiron).

In a preferred embodiment of this invention, evaluation of whether aminoacid position 236 of HIV-1 reverse transcriptase was wild type or mutantwas carried out using a phenotypic susceptibility assay using resistancetest vector DNA prepared from the biological sample. In one embodiment,plasma sample was collected, viral RNA was purified and an RT-PCRmethodology was used to amplify a patient derived segment encoding theHIV-1 protease and reverse transcriptase regions. The amplified patientderived segments were then incorporated, via DNA ligation and bacterialtransformation, into an indicator gene viral vector thereby generating aresistance test vector.

Resistance test vector DNA was isolated from the bacterial culture andthe phenotypic susceptibility assay was carried out as described inExample 1. The results of the phenotypic susceptibility assay with apatient sample having a P236L mutation. The nucleic acid (DNA) sequenceof the patient derived HIV-1 protease and reverse transcriptase regionsfrom patient sample 268 was determined using a fluorescence detectionchain termination cycle sequencing methodology (ABI/PE). The method wasused to determine a consensus nucleic acid sequence representing thecombination of sequences of the mixture of HIV-1 variants existing inthe subject sample (representing the quasispecies), and to determine thenucleic acid sequences of individual variants.

Phenotypic susceptibility profiles of patient samples and site directedmutants showed that delavirdine and nevirapine susceptibility correlatedwith the absence of RT mutations at positions 103, 181 or 236 or HIV-1reverse transcriptase. Phenotypic susceptibility profiles of patientsamples and site directed mutants snowed a significant reduction indelavirdine susceptibility (increased resistance) and little or noreduction in nevirapine susceptibility correlated with a mutation in thenucleic acid sequence encoding the amino acid leucine (L) at position236 of HIV-1 reverse transcriptase and the absence of mutations atpositions 103 and 181.

Phenotypic susceptibility profiles of patient samples and site directedmutants showed no additional reduction in delavirdine or nevirapinesusceptibility (increased resistance) with the amine acid proline atposition 236 when the RT mutations at positions 103, 181 or 103 and 181were present (K103N, Y181C, or K103N+Y181C). However, phenotypicsusceptibility profiles of patient samples and site directed mutantsshowed an additional reduction in delavirdine susceptibility (increasedresistance) and little or no additional reduction in nevirapinesusceptibility with the amino acid leucine (L) at position 236 inaddition to the RT mutations associated with NNRTI resistance (K103N,Y181C, or K103N+Y181C).

Phenotypic and Genotypic Correlation of Mutations at Amino Acid 225 ofHIV-1 Reverse Transcriptase

Phenotypic susceptibility profiles of patient samples and site directedmutants showed no change in susceptibility to delavirdine or nevirapinewhen the amino acid proline (P) was present at position 225 or HIV-1reverse transcriptase in the absence of RT mutations associated withNNRTI resistance (K103N, Y181C). However, phenotypic susceptibilityprofiles of patient samples and site directed mutants showed an increasein delavirdine susceptibility and little or no change nevirapinesusceptibility when the amino acid histidine (H) was present at position225 in the absence of RT mutations (K103N, Y181C) associated with NNRTIresistance.

Phenotypic susceptibility profiles of patient samples and site directedmutants showed no additional reduction in delavirdine susceptibility ornevirapine susceptibility when the amino acid proline (P) at position225 was present in addition to the RT mutations associated with NNRTIresistance (K103N, Y181C, or K103N+Y181C). In contrast phenotypicsusceptibility profiles of patient samples and site directed mutantsshowed an increase in delavirdine susceptibility and little or no changein nevirapine susceptibility when the amino acid histidine (H) waspresent at position 225 in the presence of RT mutations associated withNNRTI resistance (K103N, Y181C, or K103N+Y181C).

Phenotypic and Genotypic Correlation of Mutations at Amino Acid 190 ofHIV-1 Reverse Transcriptase

Phenotypic susceptibility profiles of patient samples and site directedmutants showed no change in susceptibility to delavirdine or nevirapinewhen the amino acid glycine (G) at position 190 was present in theabsence of RT mutations associated with NNRTI resistance (K103N, Y181C).Phenotypic susceptibility profiles of site directed mutants showed anincrease in delavirdine susceptibility and a decrease in nevirapinesusceptibility when the amino acid alanine (A) was present at position190 in the absence of RT mutations associated with NNRTI resistance.Phenotypic susceptibility profiles of patient samples and site directedmutants showed an increase in delavirdine susceptibility and a decreasein nevirapine susceptibility when the amino acid serine (S) was presentat position 190 in the absence of RT mutations associated with NNRTIresistance.

EXAMPLE 8 Using Resistance Test Vectors and Site Directed Mutants toCorrelate Genotypes and Phenotypes Associated with NNRTI

Drug Susceptibility and Resistance in HIV: Y181I Preparation ofResistant Test Vectors and Phenotypic Analysis of Patient 98-964 HIVSamples

A resistant test vector was constructed as described in Example 1 from apatient sample designated 98-964. This patient had been previouslytreated with ddI, d4T, AZT, 3TC, ddC, (NRTIs), saquinavir and nelfinavir(PRIs) and nevirapine (an NNRTI) and HU. Isolation of viral RNA andRT/PCR was used to generate a patient derived segment that comprisedviral sequence coding for all of PR and aa 1-313 of RT. The PDS wasinserted into an indicator gene viral vector to generate a resistancetest vector designated RTV-964. RTV-964 was then tested in a phenotypicassay to determine accurately and quantitatively the level ofsusceptibility to a panel of anti-retroviral drugs. This panel ofanti-retroviral drugs comprised members of the classes known as NRTIs(AZT, 3TC, d4T, ddI and ddC), NNRTIs (delavirdine and nevirapine), andPRIs (indinavir, nelfinavir, ritonavir, and saquinavir). An IC50 wasdetermined for the resistance test vector pool for each drug tested. Thepattern of susceptibility to all of the drugs tested was examined andcompared to known patterns of susceptibility. A pattern ofsusceptibility to NNRTIs was observed for patient RTV-964 in which therewas a moderate decrease (10-fold) in delavirdine susceptibility and asignificant decrease (750-fold) in nevirapine susceptibility.

Determination of Genetype of Patient HIV Samples

RTV-964 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database, Los Alamos, N. Mex.). Thegenotype was examined for sequences that are different from the controlsequence. Mutations were noted at positions M41L, K43E, D67N, K70R,L74I, V75S, Y181I, R211T, T215Y, D218E, and K219Q compared to thecontrol sequence. M41L, D67N, K70R, L74I, V75S, T215Y, and K21SQ areassociated with NRTI resistance. A mutation at R211T is a knownpolymorphism in the sequence among different wild-type (drug-sensitive)variants of HIV. Y181I had previously been shown to be associated withhigh level resistance to nevirapine. We examined the mutation, Y181I,using site directed mutagenesis and in vitro phenotypic susceptibilitytesting to correlate the observed changes in genotype with phenotype.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Anti-Retroviral Drugs in HIV

The Y181I mutation was introduced into the resistance test vector usingthe mega-primer method for site-directed mutagenesis (Sakar and Sommar,Ibid). A resistance test vector containing the Y181I mutation(Y181I-RTV) was then tested using the phenotypic assay described earlierand the results were compared to those determined using a geneticallydefined resistance test vector that was wild type at position 181. Wedetermined the pattern of phenotypic susceptibility to the NNRTIs,delavirdine, nevirapine and efavirenz, in the Y181I-RTV. On a wild typebackground (i.e. Y181I mutation alone) the Y181I-RTV displayed amoderate loss of susceptibility (20-fold) to delavirdine and asignificant loss of susceptibility (740-fold) to nevirapine compared toa wild type control RTV. The Y181I-RTV showed wild-type susceptibility(1.4-fold) to efavirenz.

EXAMPLE 9 Using Resistance Test Vectors and Site Directed Mutants toCorrelate Genotypes and Phenotypes Associated with NNRTI DrugSusceptibility and Resistance in HIV: Y188

Preparation of Resistant Test Vectors and Phenotypic Analysis of Patient97-300 HIV Samples

A resistance test vector was constructed as described in Example 1 froma patient sample designated 97-300. This patient had been previouslytreated with d4T and 3TC (NRTIs), indinavir (a PRI) and efavirenz (anNNRTI). Isolation of viral RNA and RT/PCR was used to generate a patientderived segment that comprised viral sequences coding for all of PR andaa 1-313 of RT. The PDS was inserted into an indicator gene viral vectorto generate a resistance test vector designated RTV-300. RTV-300 wasthen tested in a phenotypic assay to determine accurately andquantitatively the level of susceptibility to a panel of anti-retroviraldrugs. This panel of anti-retroviral drugs comprised members of theclassed known as NRTIs (AZT, 3TC, d4T, ddI and ddC), NNRTIs(delavirdine, efavirenz and nevirapine), and PRIs (indinavir,nelfinavir, ritonavir, and saquinavir). An IC50 was determined for theresistance test vector pool for each drug tested. The pattern ofsusceptibility to all of the drug tested was examined and compared toknown patterns of susceptibility. A pattern of susceptibility to theNNRTIs was observed for patient RTV-300 in which there was moderatedecrease (25-fold) in delavirdine susceptibility and a substantialdecrease (greater than 800-fold) in nevirapine susceptibility.

Determination of Genotype of Patient HIV Samples

RTV-300 DNA analyzed by ABI chain terminator automated sequencing. Thenucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database, Los Alamos, N. Mex.). Thegenotype was examined for sequence that are different from the controlsequence. Mutations were noted at positions K32N, M184V and Y188Lcompared to the control sequence. The mutation at M184V is associatedwith 3TC resistance. Y188L had previously been shown to be associatedwith high level resistance to efavirenz. Other mutations at positionY188 (i.e. Y188C and Y188H) have been reported to have been selected forby treatment with several NNRTIs (E-ePseU, E-EPS, HEPT, Nevirapine,BHAP, U-8720E, TIBO R082913, Loviride). We examined the mutation, Y188L,using site directed mutagenesis and in vitro phenotypic susceptibilitytesting to correlate the observed changes in genotype with phenotype.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Antiretroviral Drugs in HIV

The Y188L mutation was introduced into the resistance test vector usingthe mega-primer method for site-directed mutagenesis (Sakar and Sommar,Ibid.). A resistance test vector containing the Y188L mutation(Y188L-RTV) was then tested using the phenotypic assay described earlierand the results were compared to those determined using a geneticallydefined resistance test vector that was wild type at position 188. Wedetermined the pattern of phenotypic susceptibility to the NNRTIs,delavirdine, nevirapine and efavirenz, in the Y188L-RTV. On a wild typebackground (i.e. Y138L mutation alone) the Y188L-RTV displayed a slightloss of susceptibility (9-fold) to delavirdine and substantial loss ofsusceptibility (greater than 800-fold) to nevirapine and a significantloss of susceptibility (109-fold) to efavirenz compared to a wild typecontrol RTV. The approximate 100-fold loss of susceptibility toefavirenz was not as high as had been previously reported.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Antiretroviral Drugs in HIV

The Y188C mutation was introduced into the resistance test vector usingthe mega-primer method for site-directed mutagenesis (Sakar and Sommar,Ibid.). A resistance test vector containing the Y188C mutation(Y188C-RTV) was then tested using the phenotypic assay described earlierand the results were compared to those determined using a geneticallydefined resistance test vector that was wild type at position 188. Wedetermined the pattern of phenotypic susceptibility to the NNRTIs,delavirdine, nevirapine and efavirenz, in the Y188C-RTV. On a wild typebackground (i.e. Y188C mutation alone) the Y188C-RTV displayed a slightloss of susceptibility (3-fold) to delavirdine and a moderate loss ofsusceptibility (30-fold) to nevirapine and efavirenz (20-fold) comparedto a wild type control RTV.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Antiretroviral Drugs in HIV

The Y188H mutation was introduced into the resistance test vector usingthe mega-primer method for site-directed mutagenesis (Sakar and Sommai,Ibid.). A resistance test vector containing the Y188H mutation(Y188H-RTV) was then tested using the phenotypic assay described earlierand the results were compared to those determined using a geneticallydefined resistance test vector that was wild type at position 188. Wedetermined the pattern of phenotypic susceptibility to the NNRTIs,delavirdine and nevirapine, in the Y188H-RTV. On a wild type background(i.e. Y188H mutation alone) the Y188H-RTV displayed a moderate loss ofsusceptibility (3.5-fold) to nevirapine compared to a wild type controlRTV. The phenotypic susceptibility of Y188H to efavirenz was notdetermined.

EXAMPLE 10 Using Resistance Test Vectors and Site Directed Mutants toCorrelate Genotypes and Phenotypes Associated with NNRTI DrugSusceptibility and Resistance in HIV: E138 and Y188

Preparation of Resistant Test Vectors and Phenotypic Analysis of Patient97-209 HIV Samples

A resistance test vector was constructed as described in Example 1 froma patient sample designated 97-209. This patient had been previouslytreated with AZT, ddI, d4T and 3TC (NRTIs), indinavir (a PRIs) andadefovir. Isolation of viral RNA and RT/PCR was used to generate apatient derived segment that comprised viral sequences ceding for all ofPR and aa 1-313 of RT. The PDS was inserted into an indicator gene viralvector to generate resistance test vector designated RTV-209. RTV-209was then tested in a phenotypic assay to determine accurately andquantitatively the level of susceptibility to a panel of anti-retroviraldrugs. This panel of anti-retroviral drugs comprised members of theclasses known as NRTIs (AZT, 3TC, d4T, ddI and ddC), NNRTIs(delavirdine, efavirenz and nevirapine), and PRIs (indinavir,nelfinavir, ritonavir, and saquinavir). An IC50 was determined for theresistance test vector pool for each drug tested. The pattern ofsusceptibility to ail of the drugs tested was examined and compared toknown patterns of susceptibility. A pattern of susceptibility to theNNRTIs was observed for patient RTV-209 in which there was a moderatedecrease (75-fold) in delavirdine susceptibility and a substantialdecrease (greater than 800-fold) in nevirapine susceptibility.

Determination of Genotype of Patient HIV Samples

RTV-209 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database, Los Alamos, N. Mex.). Thegenotype was examined for sequences that are different from the controlsequence. Mutations were noted at positions A62V, S68G, V761, F77L,F116Y, E138A, Q151M, M184V, Y188L and E291D compared to the controlsequence. The mutations at A62V, V751, F77L, F116Y, Q151M and M184V areassociated with NRTI resistance. A mutation at E138K had previously beenshown to be associated with resistance to several NNRTIs and a mutationat Y188L had previously been shown to be associated with a decrease insusceptibility to efavirenz. We examined the mutations Y188L and E136Ausing site directed mutagenesis and in vitro phenotypic susceptibilitytesting to correlate the observed changes in genotype with phenotype.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Antiretroviral Drugs in HIV

The E138A mutation alone and in combination with Y188L was introducedinto resistance test vectors using the mega-primer method forsite-directed mutagenesis (Sakar and Sommar, Ibid.). Resistance testvectors containing the E138A mutation (E138A-RTV) or the E138 mutationalong with the Y188L mutation (E138A-Y188L-RTV) were then tested usingthe phenotypic assay described earlier and the results were compared tothose determined using a genetically defined resistance test vector thatwas wild type at positions 188 and 138. We determined the pattern ofphenotypic susceptibility to the NNRTIs, delavirdine, nevirapine andefavirenz, in the E138A-RTV, Y188L-RTV and E138-Y188L-RTV. On a wildtype background (i.e. E138A mutation alone) the E138A-RTV displayedwild-type susceptibility to delavirdine (1.6-fold), nevirapine(1.3-fold) and efavirenz (1.4-fold). The Y188L-RTV displayed a slightloss of susceptibility (greater than 800-fold) to nevirapine and asignificant loss of susceptibility (110-fold) to efavirenz. TheE138A-Y188L-RTV displayed a moderate loss of susceptibility (75-fold) todelavirdine and efavirenz (88-fold) and a substantial loss ofsusceptibility to nevirapine (greater than 800-fold) compared to a wildtype control RTV. The combination of mutations resulted in an increasedeffect on delavirdine susceptibility compared to the effect observed foreach mutation alone.

EXAMPLE 11 Using Resistance Test Vectors and Site Directed Mutants to 2Correlate Genotypes and Phenotypes Associated with NNRTI DrugSusceptibility and Resistance in HIV: A98

Preparation of Resistant Test Vectors and Phenotypic Analysis of Patient98-675 HIV Samples

A resistance test vector was constructed as described in Example 1 froma patient sample designated 98-675. This patient had been previouslytreated with ddI, AZT, and 3TC (NRTIs), and saquinavir and nelfinavir(PRIs). Isolation of viral RNA and RT/PCR was used to generate a patientderived segment that comprised viral sequences coding for all of PR andaa 1-313 of RT. The PDS was inserted into an indicator gene viral vectorto generate a resistance test vector designated RTV-765. RTV-675 wasthen tested in a phenotypic assay to determine accurately andquantitatively the level of susceptibility to a panel of anti-retroviraldrugs. This panel of anti-retroviral drugs comprised members of theclasses known as NRTIs (AZT, 3TC, d4T, ddI and ddC), NNRTIs(delavirdine, efavirenz and nevirapine), and PRIs (indinavir,nelfinavir, ritonavir, and saquinavir). An IC50 was determined for theresistance test vector pool for each drug tested. The pattern ofsusceptibility to all of the drugs tested was examined and compared toknown patterns of susceptibility. A pattern of susceptibility to theNNRTIs was observed for patient RTV-675 in which wild-typesusceptibility (2.1-fold) was observed for delavirdine and a slightdecrease (6-fold) in nevirapine susceptibility was observed.

Determination of Genotype of Patient HIV Samples

RTV-675 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database, Los Alamos, N. Mex.). Thegenotype was examined for sequences that are different from the controlsequence. Mutations were noted at positions M41L, S48t, L74V, A98G,M184V and T215Y are associated with NRTI resistance. A mutation at A98Ghad previously been shown to be associated with resistance tonevirapine. We examined the mutation A98G using site directedmutagenesis and in vitro henotypic susceptibility testing to correlatethe observed changes in genotype with phenotype.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Antiretroviral Drugs in HIV

The A98G mutation into the resistance test vector using the mega-primermethod for site-directed mutagenesis (Sakar and Sommar, Ibid.). Aresistance test vector containing the A98G mutation (A98G-RTV) was thentested using the phenotypic assay described earlier and the results werecompared to those determined using a genetically defined resistance testvector that was wild type at position 98. We determined the pattern ofphenotypic susceptibility to the NNRTIs, delavirdine, nevirapine andefavirenz, in the A98G-RTV. On a wild type background (i.e. A98Gmutation alone) the A98G RTV displayed a slight loss of susceptibilityto delavirdine (3-fold), nevirapine (8-fold) and efavirenz (3-fold)compared to a wild type control RTV.

EXAMPLE 12 Using Resistance Test Vectors and Site Directed Mutants toCorrelate Genotypes and Phenotypes Associated with NNRTI DrugSusceptibility and Resistance in HIV: A98 and G190

Preparation of Resistant Test Vectors and Phenotypic Analysis of PatientB HIV Samples.

A resistant test vector was constructed as described in Example 1 from apatient sample designated B. The anti-retroviral treatment this patientreceived is unknown. Isolation of viral RNA and RT/PCR was used togenerate a patient derived segment that comprised viral sequences codingfor ail of PR and aa 1-313 of RT. The PDS was inserted into an indicatorgene viral vector to generate a resistant test vector designated RTV-B.Individual clones of the RTV-B pool were selected and then tested in aphenotypic assay to determine accurately and quantitatively the level ofsusceptibility to a panel of anti-retroviral drugs. This panel ofanti-retroviral drugs comprised members of the classes known as NRTIs(AZT, 3TC, d4T, ddI and ddC), NNRTIs (delavirdine and nevirapine), andPRIs (indinavir, nelfinavir, ritonavir, and saquinavir). An IC50 wasdetermined for the resistance test vector clone for each drug tested.The pattern of susceptibility to all of the drugs tested was examinedand compared to known patterns of susceptibility. A pattern ofsusceptibility to the NNRTIs was observed for patient RTV-3 clone 1 inwhich there was an increase in susceptibility (0.55-fold) todelavirdine, a substantial loss of susceptibility (640-fold) tonevirapine and significant loss of susceptibility (250-fold) toefavirenz.

Determination of Genotype of Patient HIV Samples

RTV-B clone 1 DNA was analyzed by ABI chain terminator automatedsequencing. The nucleotide sequence was compared to the consensussequence of a wild type clade B HIV-1 (HIV Sequence Database, LosAlamos, N. Mex.). The genotype was examined for sequences that aredifferent from the control sequence. Mutations were noted at positionsM41L, A98G, M184V, L210W, R211?, T215Y, E397 and G190S compared to thecontrol sequence M41L, M184V, L210W and T215Y are associated with NRTIresistance. A mutation at A98G had previously been shown to beassociated with resistance to nevirapine. A mutation at position G190Ahad previously been shown to be associated with changes insusceptibility to nevirapine. Other changes at position 190 (i.e. E, Q,and T) have also been reported. We examined the mutations A98G andG190S, using site directed mutagenesis and in vitro phenotypicsusceptibility testing to correlate the observed changes in genotypewith phenotype.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Antiviral Drugs in HIV

The A98 and G190S mutations were introduced alone or in combination intothe resistance test vector using the mega-primer method forsite-directed mutagenesis (Sakar and Sommar, Ibid.). Resistance testvectors containing the A98G mutation (A98G-RTV), the G190S mutation(G190S-RTV) and both mutations (A98G-G190S-RTV) were then tested usingthe phenotypic assay described earlier and the results were compared tothose determined using a genetically defined resistance test vector thatwas wild type at position 98 and 190. We determined the pattern ofphenotypic susceptibility to the NNRTIs, delavirdine, nevirapine andefavirenz, in the three vectors. On a wild type background (i.e. A98Gmutation alone) the A98G-RTV displayed a slight loss of susceptibilityto delavirdine (3-fold), nevirapine (8-fold) and efavirenz (3-fold)compared to a wild type control RTV. On a wild type background (i.e.G190S mutation alone) the G190S-RTV displayed increased susceptibility(0.5-fold) to delavirdine, a moderate loss of susceptibility (75-fold)to nevirapine and a slight loss of susceptibility (8-fold) to efavirenzcompared to a wild type control RTV. The A98G-G190S-RTV displayedincreased susceptibility (0.8-fold) to delavirdine, but a substantialloss of susceptibility to both nevirapine greater than 300-fold) andefavirenz (greater than 250-fold) compared to a wild type control RTV.Although only a slight loss of susceptibility to efavirenz was observedfor the individual mutations, the combination of A98G and G190S resultedm a substantial loss of susceptibility to efavirenz. Likewise, thiscombination of mutation resulted in a greater loss of susceptibility tonevirapine than the sum of the two mutations alone.

EXAMPLE 13 Using Resistance Test Vectors and Site Directed MutantsCorrelate Genotypes and Phenotypes Associated with NNRTI DrugSusceptibility and Resistance in HIV: Y181 and A98

Preparation of Resistant Test Vectors and Phenotypic Analysis of Patient98-1057 Samples

A resistance test vector was constructed as described in Example 1 froma patient sample designated 98-1057. This patient had been previouslytreated with ddI, d4T, AZT, and 3TC (NRTIs), saquinavir and indinavir(PRIs) and delavirdine (an NNRTI). Isolation of viral RNA and RT/PCR wasused to generate a patient derived segment that comprised viralsequences coding for all of PR and aa 1-313 RT. The PDS was insertedinto an indicator gene viral vector to generate resistance test vectordesignated RTV-1057. RTV-1057 was then tested in a phenotypic assay todetermine accurately and quantitatively the level or susceptibility to apanel of anti-retroviral drugs. This ranel of anti-retroviral drugscomprised members of the classes known as NRTIs (AZT, 3TC, d4T, ddI, andddC), NNRTIs (delavirdine, efavirenz and nevirapine) and PRIs(indinavir, nelfinavir, ritonavir, and saquinavir). An IC50 wasdetermined for the resistance test vector pool for each drug tested. Thepattern of susceptibility to all of the drugs tested was examined andcompared to known patterns of susceptibility. A pattern ofsusceptibility to the NNRTIs was observed for patient RTV-1057 in whichthere was a moderate decrease in delavirdine (35-fold) susceptibilityand a significant decrease (610-fold) in nevirapine susceptibility.

Determination of Genotype of Patient HIV Samples

RTV-1057 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide frequency was compared to the consensus sequence of awild type clade B HIV-1 (HIV Sequence Database, Los Alamos, N. Mex.).The genotype was examined for sequences that are different from thecontrol sequence. Mutations were noted at positions T39A, M41L, A62V,D67E, T69SST, A98G, I135T, Y181C, T200I and T215Y compared to thecontrol sequence M41L, A62V, D67E, T69SST, and T215Y are associated withNRTI resistance. Mutations at positions I135T and T200I are knownpolymorphisms in the sequence among different wild-type (drug-sensitive)variants of HIV. Y181C and A98G have been previously shown to beassociated with resistance to certain NNRTIs. We examined the mutationsY181C and A98G using site directed mutagenesis and in vitro phenotypicsusceptibility testing to correlate the observed changes in genotypewith phenotype.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Anti-Retroviral Drugs in HIV

The Y181C and A98G mutations were introduced alone and in combinationinto resistance test vectors using the mega-primer method forsite-directed mutagenesis (Sakar and Sommar, Ibid.). Resistance testvectors containing the Y181C mutation (Y131C-RTV) and the A98G mutation(A98G-RTV) and both mutations (Y181C-A98G-RTV) were then tested usingthe phenotypic assay described earlier and the results were compared tothose determined using a genetically defined resistance test vector thatwas wild type at position 181 and 98. We determined the pattern ofphenotypic susceptibility to the NNRTIs, delavirdine, nevirapine andefavirenz, in the three vectors. On a wild type background (i.e. Y181Cmutation alone) the Y181C-RTV displayed moderate loss of susceptibility(35-fold) to delavirdine, a significant loss of susceptibility(161-fold) to nevirapine and a slight loss of susceptibility (3-fold) toefavirenz compared to a wild type control RTV. The A98G-RTV displayed aslight loss of susceptibility to delavirdine (3-fold), nevirapine(8-fold) and efavirenz (3-fold) compared to a wild type control RTV. TheY181C-A98G-RTV displayed significant loss of susceptibility (240-fold)to delavirdine, a substantial loss of susceptibility (greater than800-fold) to nevirapine and a slight loss of susceptibility (7-fold) toefavirenz compared to a wild type control RTV. These data indicated thatthe combination of the two mutations, Y181C and A98G, resulted in agreater loss of susceptibility to both delavirdine and nevirapine thanthe sum of effects observed for these two mutations individually.

EXAMPLE 14 Using Resistant Test Vectors and Site Directed Mutants toCorrelate Genotypes and Phenotypes Associated with NNRTI DrugSusceptibility and Resistance in HIV: K101 and G190

Preparation of Resistant Test Vectors and Phenotypic Analysis ofPatients 98-644 and 98-1060 HIV Samples

A resistance test vector was constructed as described in Example 1 froma patient sample designated 98-644. This patient had been previouslytreated with d4T (an NNRTI), indinavir (a PRI) and efavirenz (an NNRTI).A second resistance test vector was constructed as described in Example1 from a patient sample designated 98-1060. This patient had beenpreviously treated with d4T (an NNRTI), indinavir (a PRI) and efavirnez(an NNRTI). Isolation of viral RNA and RT/PCR was used to generate apatient derived segment that comprised viral sequences coding for all ofPR and aa 1-313 of RT. The PDS was inserted into an indicator gene viralvector to generate resistance test vectors designated RTV-644 andRTV-1060. RTV-644 and RTV-1060 were then tested in a phenotypic assay todetermine accurately and quantitatively the level or susceptibility to apanel of anti-retroviral drugs. This panel of anti-retroviral drugscomprised members of the classes known as NNRTIs (AZT, 3TC, d4T, ddI,and ddC), NNRTIs (delavirdine and nevirapine), and PRIs (indinavir,nelfinavir, ritonavir, and saquinavir). An IC50 was determined for theresistance test vector pool for each drug tested. The pattern ofsusceptibility to all of the drugs tested was examined and compared toknown patterns of susceptibility. A pattern of susceptibility to theNNRTIs was observed for patient RTV-644 in which there was a very slight(2.5-fold) decrease in delavirdine susceptibility and a significant(600-fold) decrease in nevirapine susceptibility. A pattern ofsusceptibility to the NNRTIs was observed for patient RTV-644 in whichthere was a very slight (2.5-fold) decrease in delavirdinesusceptibility and a significant (600-fold) decrease in nevirapinesusceptibility. A pattern of susceptibility to the NNRTIs was observedfor patient RTV-1060 in which wold-type susceptibility (1.5-fold) todelavirdine was observed. A significant decrease in efavirenzsusceptibility (900-fold) and a substantial decrease to nevirapine(greater than 800-fold) susceptibility was observed for RTV-1060.

Determination of Genotype of Patient HIV Samples

RTV-644 and RTV-1060 DNA were analyzed by ABI chain terminator automatedsequencing. The nucleotide sequence was compared to the consensus of awild type clade B HIV-1 (HIV Sequence Database, Los Alamos, N. Mex.).The genotype was examined for sequences that are different from thecontrol sequence. Mutations were noted at positions K101E and G190S forRTV-644 compared to the control sequence and mutations were noted atpositions K101E, G190S, T200A and T215Y for RTV-1060 compared to thecontrol sequence. The sequence at position T215 was a mixture ofwild-type and mutation. A mutation at position K101E had been previouslyshown to be associated with resistance to several NNRTIs including highlevel resistance to delavirdine. A mutation at position G190A hadpreviously been shown to be associated with changes in susceptibility tonevirapine. Other changes at position 190 (i.e. E, C and T) have alsobeen reported. We examined the mutations K101E and G190S, using sitedirected mutagenesis and in vitro phenotypic susceptibility testing tocorrelate the observed changes in genotype with phenotype.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Antiretroviral Drugs in HIV

The K101E and G190S mutations were introduced alone and in combinationinto resistance test vectors using the megaprimer method forsite-directed mutagenesis (Sakar and Sommar, Ibid.). Resistance testvectors containing the K101E mutation (K101E-RTV), the G190S mutation(G190S-RTV) were then tested using the phenotypic assay describedearlier and the results were compared to those determined using agenetically defined resistance test vector that was wild type atpositions 101 and 190. We determined the pattern of phenotypicsusceptibility to the NNRTIs, delavirdine, nevirapine and efavirenz, inall three vectors. On a wild type background (i.e. K101E mutation alone)the K101E-RTV displayed a slight loss of susceptibility (5-fold) todelavirdine and efavirenz (5-fold) and a moderate loss of susceptibility(12-fold) to nevirapine compared to a wild type control RTV. TheK101E-G190S-RTV displayed increased susceptibility to delavirdine(0.5-fold), a moderate loss of susceptibility to nevirapine (75-fold)and a slight loss of susceptibility (7.6-fold) to efavirenz compared toa wild type control RTV. The K101E-G190S-RTV displayed wild-typesusceptibility (1.4-fold) to delavirdine and a substantial loss ofsusceptibility; o both nevirapine (greater than 800-fold) and efavirenz(greater than 250-fold) compared to a wild type control RTV.

In this example, the combination of mutations, G190S and K101E,displayed a novel phenotypic pattern. The combination resulted in thereversal of the effect on delavirdine susceptibility observed for theG190S mutation alone and a greater than additive effect on thesusceptibility for both nevirapine and efavirenz.

EXAMPLE 15 Using Resistance Test Vectors and Site Directed Mutants toCorrelate Genotypes and Phenotypes Associated with NNRTI DrugSusceptibility and Resistance in HIV: V108I

Preparation of Resistant Test Vectors and Phenotypic Analysis of Patient98-652 HIV Samples

A resistance test vector was constructed as described in Example 1 froma patient sample designated 98-652. This patient had no previousanti-retroviral treatment. Isolation of viral RNA and RT/PCR was used togenerate a patient derived segment that comprised viral sequences codingfor all of PR and aa 1-313 or RT. The PDS was inserted into an indicatorgene viral vector to generate a resistance test vector designatedRTV-652. RTV-652 was then tested in a phenotypic assay to determineaccurately and quantitatively the level of susceptibility to a panel ofanti-retroviral drugs. This panel of anti-retroviral drugs comprisedmembers of the classes known as NRTIs (AZT, 3TC, d4T, ddI and ddC),NNRTIs (delavirdine and nevirapine), and PRIs (indinavir, nelfinavir,ritonavir and saquinavir). An IC50 was determined for the resistancetest vector pool for each drug tested. The pattern of susceptibility toall of the drugs tested was examined and compared to known patterns ofsusceptibility. A pattern of susceptibility to the NNRTIs was observedfor patient RTV-652 in which increase susceptibility (0.97-fold) todelavirdine was observed and a slight decrease (5-fold) in nevirapinesusceptibility was observed.

Determination of Genotype of Patient HIV Samples

RTV-652 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV Sequence Database, Los Alamos, N. Mex.). Thegenotype was examined for sequences that are different from the controlsequence. Mutations were noted at positions M41L, V108I, I135T, L210W,R21K and T215D compared to the control sequence. M41L, L210W and T215Dare associated with NRTI resistance. Mutations at positions I135T andR211K are known polymorphisms in the sequence among different wild-type(drug-sensitive) variants of HIV. V108I is known to be associated withresistance to several NNRTIs. We examined the mutation V108I using sitedirected mutagenesis and in vitro phenotypic susceptibility testing tocorrelate the observed changes in genotype with phenotype.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Antiretroviral Drugs in HIV

The V108I mutation was introduced into the resistance test vector usingthe mega-primer method for site directed mutagenesis (Sakar and Sommar,Ibid.). A resistance test vector containing the V108I mutation(V108I-RTV) was then tested using the phenotypic assay described earlierand the results were compared to those determined using a geneticallydefined resistance test vector that was wild type at position 108. Wedetermined the pattern of phenotypic susceptibility to the NNRTIs,delavirdine, nevirapine and efavirenz, in the V108I-RTV. On a wild typebackground (i.e. V108I mutation alone) the V108I-RTV displayed wild-typesusceptibility (1.3-fold) to delavirdine and efavirenz (1.7-fold) and aslight loss of susceptibility (13-fold) to nevirapine compared to a typecontrol RTV.

EXAMPLE 16 Using Resistance Test Vectors and Site Directed Mutants toCorrelate Genotypes and Phenotypes Associated with NNRTI

Drug Susceptibility and Resistance in HIV: K103 and K101 and G190

Preparation of Resistant Test Vectors and Phenotypic Analysis of Patient98-955 HIV Samples

A resistance test vector was constructed as described in Example 1 froma patient sample designated 98-955. This patient had been previouslytreated with nelfinavir (a PRI). Isolation of viral RNA and RT/PCR wasused to generate a patient derived segment that comprised viralsequences coding for all of PR and aa 1-313 of RT. The PDS was insertedinto an indicator gene viral vector to generate a resistance testvectors designated RTV-955. RTV-955 was then tested in a phenotypicassay to determine accurately and quantitatively the level ofsusceptibility to a panel of anti-retroviral drugs. This panel ofanti-retroviral drugs comprised members of the classes known as NRTIs(AZT, 3TC, d4T, ddI and ddC), NNRTIs (delavirdine, efavirenz andnevirapine), and PRIs (indinavir, nelfinavir, ritonavir, and aquinavir).An IC50 was determined for the resistance test vector pool for each drugtested. The pattern of susceptibility to all of the drugs tested wasexamined and compared to known patterns of susceptibility. A pattern ofsusceptibility to the NNRTIs was observed for patient RTV-955 in whichthere was a slight decrease (4-fold) in delavirdine susceptibility and asignificant decrease (530-fold) in nevirapine susceptibility.

Determination of Genotype of Patient HIV Samples

RTC-955 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of wildtype clade B HIV-1 (HIV Sequence Database, Los Alamos, N. Mex.). Thegenotype was examined for sequences that are different from the controlsequence. Mutations were noted it positions K20R, V35I, A62V, D67N,T69D, 7751, F77L, K101E, X103N, Y115F, F116Y, Q151M, I167V, Y181C,M184V, G190A, I202V, R211K, F214L, T215V, and K219Q compared to thecontrol sequence. Mutations at positions K101E, K103N, Y181C, G190A, andF214L were mixtures of wild-type and the mutation. A62V, D67N, T69D,V751, F77L, Y115F, F116Y, Q151M, M184V, T215V and K219Q are associatedwith NRTI resistance. Mutations at V35I, R211K and F214L are knownpolymorphism in the sequence among different wild-type (drug sensitive)variants of HIV. A mutation at position K101E had been previously shownto be associated with resistance to the NNRTIs. A mutation at Y181I hadpreviously been shown to be associated with high level resistance tonevirapine. A mutation at K103N had previously been shown to beassociated with resistance to the three NNRTIs, delavirdine andnevirapine and efavirenz. We examined the mutations K101E, J103N andG190A using site directed mutagenesis and in vitro phenotypicsusceptibility testing to correlate the observed changes in genotypewith phenotype.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Anti-Retroviral Drugs in HIV

The K101E, K103N and G190A mutations were introduced alone and incombination inco resistance test vectors using the mega-primer methodfor site-directed mutagenesis (Sakar and Sommar, Ibid.). Resistance testvectors containing the K101E mutation (K101E-RTV), the K103N mutation(K103N-RTV), the G190 mutation (G190A-RTV) and two mutations(K101E-G190A-RTV) and (K103N-G190A-RTV) were then tested using thephenotypic assay described earlier and the results were compared tothose determined using a genetically defined resistance test vector thatwas wild type at positions 101, 103 and 190. We determined the patternof phenotypic susceptibility to the NNRTIs, delavirdine, nevirapine, andefavirenz, in all 5 vectors. On a wild type background (i.e. K101Emutation alone) the K101E-RTV displayed a slight loss (5-fold) ofsusceptibility to delavirdine and efavirenz (5-fold) and a moderate lossof susceptibility (12-fold) to nevirapine (55-fold) and efavirenz(30-fold) compared to a wild type control RTV. On a wild type background(i.e. G190A mutation alone) the G190A-RTV displayed increasedsusceptibility (8-fold) efavirenz compared to a wild type control RTV.The K101E-G190A-RTV displayed wild-type susceptibility (2-fold) todelavirdine, substantial loss of susceptibility (greater than 800-fold)to nevirapine and a significant loss of susceptibility (120-fold) toefavirenz compared to a wild type control RTV. The K103N-G190-RTVdisplayed a moderate loss of susceptibility (40-fold) to delavirdine,substantial loss of susceptibility (greater than 800-fold) to nevirapineand a significant loss of susceptibility (215-fold) to efavirenzcompared to a wild type control RTV. The introduction of a secondmutation to a vector containing the G190A resulted in the reversal ofthe effect on delavirdine susceptibility observed for the G190A mutationalone. The G190-a mutation displayed an increased susceptibility todelavirdine. However, the addition of either K101E or K103N to the G190Amutation resulted in s slight loss of susceptibility to delavirdine.Furthermore, the combination of G190A and K101E resulted in a greaterthan additive effect on the loss of susceptibility to nevirapine andefavirenz. Lastly, these data indicated that the combination of the twomutations G190A and K103N resulted in a greater loss of susceptibilityto both nevirapine and efavirenz than the sum of effects observed forthese two mutations individually.

EXAMPLE 17 Using Test Vectors and Site Directed Mutants to CorrelateGenotypes and Phenotypes Associated with NNRTI Drug Susceptibility andResistance in HIV: V106 and V189 and V181 and F227

Preparation of Resistant Test Vectors and Phenotypic Analysis of Patient98-1033 and 98-757 HIV Samples

A resistance test vector was constructed as described in Example 1 froma patient sample designated 98-1033. This patient had been previouslytreated with AZT, d4T, 3TC and ddI (NRTI), saquinavir, indinavir andnelfinavir (PRIs and nevirapine (an NNRTI)). A second resistance testvector was constructed as described in Example 1 from a sample obtainedfrom the same patient at a different time point and designated 98-757.This patient had received an additional 8 weeks of treatment withnevirapine (an NNRTI) d4T (an NRTI), and saquinavir and nelfinavir(PRIs). Isolation of viral RNA and RT/PCR was used to generate a patientderived segment that comprised viral sequences coding for all of PR andaa 1-313 of RT. The PDS was inserted into an indicator gene viral vectorto generate resistance test vectors designated RTV-1033 and RTV-757.RTV-1033 and RTV-757 were then tested in a phenotypic assay to determineaccurately and quantitatively the level of susceptibility to a panel ofanti-retroviral drugs. This panel of anti-retroviral drugs comprisedmembers of the classes known as NRTIs (AZT, 3TC, d4T, ddI and ddC),NNRTIs (delavirdine and nevirapine), and PRIs (indinavir, nelfinavir,ritonavir, and saquinavir). An IC50 was determined for the resistancetest vector pool for each drug tested. The pattern of susceptibility toall if the drugs tested was examined and compared to known patterns ofsusceptibility. A pattern of susceptibility to the NNRTIs was observedfor patient RTV-1033 in which there was a moderate decrease (30-fold) indelavirdine susceptibility and a substantial decrease (greater than800-fold in nevirapine susceptibility and a significant decrease(200-fold) in efavirenz susceptibility. A pattern of susceptibility tothe NNRTIs was observed for patient RTV-757 in which there was a slightdecrease (10-fold) in delavirdine susceptibility and a substantialdecrease (greater than 300-fold) in nevirapine susceptibility.

Determination of Genotype of Patient HIV Samples

RTV-1033 and RTV-757 DNA were analyzed by ABI chain terminator automatedsequencing. The nucleotide sequence was compared to the consensussequence of a wild type clade B HIV-1 (HIV Sequence Database, LosAlamos, N. Mex.). The genotype was examined for sequences that aredifferent from the control sequence. Mutations were noted at positionsV35I, D67N, T69D, K70R, V106A, V189L, T200A, I202T, R211K, T215F, D218E,K219Q, H221Y, F227L, L228H and R284 for RTV-1033 compared to the controlsequence. Mutations were noted at positions V35I, D67N, T69D, K70R,V106A, V108I, L109V, Y108C, V189L, T200A, I202T, R211K, T215F, D218E,K219Q, H221Y, L228H, L283I and R284K for RTV-757 compared to the controlsequence. The sequences at positions V106A, V108I and L109V were amixture of wild-type and mutation. D67N, T69D, K70R, T215F and K219Q areassociated with NRTI resistance. Mutations at 735I, T200A, R211K andR284K are known polymorphisms in the sequence among different wild-typedrug-sensitive) variants of HIV. A mutation at V106A had previously beenshown to be associated with increased resistance to nevirapine. Amutation at V189I had previously been shown to be associated with NNRTIresistance but a mutation to L. at this position had not been previouslyreported to be associated with NNRTI resistance. A mutation at V108I hadpreviously been shown to be associated with increased resistance to bothdelavirdine and nevirapine. A mutation at Y181C had also previously beenshown to be associated with increased resistance to both delavirdine andnevirapine. We examined the mutations V106A, V189L, V181C and F227Lusing site directed mutagenesis and in vitro phenotypic susceptibilitytesting to correlate the observed changes in genotype with phenotype.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Anti-Retroviral Drugs in HIV

The mutations V106A, V189L, V181C an F227L were introduced alone and incombination into resistance test vectors using the mega-primer methodfor site-directed mutagenesis (Sakar and Sommar, Ibid.). Resistance testvectors containing the VI 06A mutation (V106A-RTV), the V189L mutation(V189L-RTV), the V181C mutation (V181C-RTV) and F227L mutation(F227L-RTV) and two mutations (V106A-Y181C-RTV) and (V106A-V189L-RTV)and (V106A-F227-RTV) and (V181C-F227-RTV) and three mutations,(V106A-Y181C-F227L-RTV) were then tested using the phenotypic assaydescribed earlier and the results were compared to those determinedusing a genetically defined resistance rest vector that was wild type atpositions 106, 189, 181 and 227. We determined the pattern of phenotypicsusceptibility to the NNRTIs, delavirdine, nevirapine and efavirenz, inall nine vectors. On a wild type background (i.e. V106A mutation alone)the V106A-RTV displayed a slight loss (5-fold) of susceptibility todelavirdine and a moderate loss of susceptibility (60-fold) tonevirapine and wild-type susceptibility (1.7-fold) to efavirenz comparedto a wild type control RTV. On a wild type background (i.e. V189Lmutation alone the V189-RTV displayed wild type susceptibility todelavirdine (1.8-fold), nevirapine (1.3-fold) and efavirenz (1.3-fold);compared to a wild type control RTV. On a wild type background (i.e.V181C mutation alone) the Y181C-RTV displayed a significant loss ofsusceptibility (100-f old) to delavirdine and a substantial loss ofsusceptibility (greater than 800-fold) to nevirapine and a slight lossof susceptibility (4-fold) to efavirenz compared to a wild type controlRTV. On a wild type background (i.e. F227L mutation alone) the F227L-RTVdisplayed increased susceptibility (0.03-fold) to delavirdine andefavirenz (0.48-fold) and a slight loss of susceptibility (3-fold) tonevirapine compared to a wild type control RTV. The V106A-Y181C-RTVdisplayed a significant loss of susceptibility (100-fold) todelavirdine, a substantial loss of susceptibility (greater than800-fold) to nevirapine and slight loss of susceptibility (4-fold) toefavirenz compared to a wild type control RTV. The V106A-V189L-RTVdisplayed a slight loss of susceptibility (3-fold) to delavirdine, amoderate loss of susceptibility (50-fold) to nevirapine and wild-typesusceptibility (1-fold) to efavirenz compared to a wild type controlRTV. The V106A-F227-RTV displayed a slight loss of susceptibility(3-fold) to delavirdine, a substantial loss of susceptibility (greaterthan 800-fold) to nevirapine and a slight loss of susceptibility(8-fold) to efavirenz compared to a wild type control RTV. TheY181C-F227L-RTV displayed increased susceptibility (0.89-fold) todelavirdine and efavirenz (0.99-fold) and a significant loss ofsusceptibility (285-fold) to nevirapine compared to a wild type controlRTV. The V106A-Y181C-F227L-RTV displayed a moderate loss (50-fold) ofsusceptibility to delavirdine and a substantial loss of susceptibility(greater than 800-fold) to nevirapine and a slight loss ofsusceptibility (12-fold) to efavirenz compared to a wild type controlRTV.

EXAMPLE 18 Using Resistance Test Vectors and Site Directed Mutants toCorrelate Genotypes and Phenotypes Associated with NNRTI DrugSusceptibility and Resistance in HIV: Y188 and L100 and K103

Preparation of Resistance Test Vectors and Phenotypic Analysis ofPatient 98-1058 HIV Samples

A resistance test vector v.-as constructed as described in Example 1from a patient sample designated 98-1058. This patient had beenpreviously treated with ddI, d4T, AZT, 3TC, ddC and abacavir (NRTIs),indinavir and amprenavir (PRIs) and nevirapine (an NNRTI). Isolation ofviral RNA and RT/PCR was used to generate a patient derived segment thatcomprised viral sequences coding for all of RP and aa 1-313 of RT. ThePDS was inserted into an indicator gene viral vector to generate aresistance test vector designated RTV-1058. Individual clones ofRTV-1058 were selected and were then tested in a phenotypic assay todetermine accurately and quantitatively the level of susceptibility to apanel of anti-retroviral drugs. The panel of anti-retroviral drugscomprised members of the classes known as NRTIs (AZT, 3TC, d4T, kkI andddC), NNRTIs (delavirdine and nevirapine), and PRIs (indinavir,nelfinavir, ritonavir, and saquinavir). An IC50 was determined for theresistance test vector pool for each drug tested. The pattern ofsusceptibility to all or the drugs tested was examined and compared toknown patterns of susceptibility. A pattern of susceptibility to theNNRTIs was observed for clones 4, and from patient RTV-1058. Clone 4displayed a significant loss of susceptibility (85-fold) for delavirdineand a substantial loss of susceptibility (greater than 800-fold) fornevirapine. Clone displayed a substantial loss of susceptibility(150-fold) to delavirdine and a significant loss of susceptibility(120-fold) to nevirapine. Clone displayed a substantial loss ofsusceptibility (greater than 250-fold) to delavirdine and (greater than800-fold) to nevirapine.

Determination of Genotype of Patient HIV Samples

RTV-1058 DNA was analyzed by ABI chain terminator automated sequencing.The nucleotide sequence was compared to the consensus sequence of a wildtype clade B HIV-1 (HIV sequence Database, Los Alamos, N. Mex.). Thegenotype was examined for sequences that are different from the controlsequence. Mutations were noted as positions M41L, A62V, D67N, T69SST,L74V, L100I, K103N, V181I, I135T, T200S, L210W, R211K and T215Y comparedto the control sequence. L74V and L100I were mixtures of wild-type andmutation. Clone 4 contained mutations at positions K103N. Clonecontained mutations at positions L100I and K103N. Clone containedmutations at positions L100I, K103N and Y188L. M41L, A62V, D67N, T69SST,L74V, L210W and T215Y are associated with NRTI resistance. Mutations atpositions I135T, T200S and R211T are known polymorphisms in the sequenceamong different wild-type (drug-sensitive) variants of HIV. A mutationat L100I had previously been shown to be associated with resistance todelavirdine and nevirapine. A mutation at K103N had previously beenshown to be associated with resistance to delavirdine, nevirapine andefavirenz. We examined the mutations, Y188L, L100I and K103N, using sitedirected mutagenesis and in vitro phenotypic susceptibility testing tocorrelate the observed changes in genetype with phenotype.

Site Directed Mutagenesis is Used to Confirm the Role of SpecificMutations in Phenotypic Susceptibility to Anti-Retroviral Drugs in HIV

The mutations Y188L, L100I and K103N were introduced alone and incombination into resistance test vectors using the mega-primer methodfor site-directed mutagenesis (Sakar and Sommar, Ibid.). Resistance testvectors containing the Y188L mutation (K103N-RTV), the two mutations(K103N-Y188L-RTV) and (L100I-K103N-RTV), and the three mutations(L100I-K103N-Y188L-RTV) were then tested using the phenotypic assaydescribed earlier and tie results were compared to those determinedusing a genetically defined resistance test vector that was wild type atpositions 188, 100, and 103. We determined the pattern of phenotypicsusceptibility to the NNRTIs, delavirdine, nevirapine and efavirenz, inall 6 vectors. On a wild type background (i.e. Y188L mutation alone) theY188L-RTV displayed a slight loss of susceptibility (9-fold) todelavirdine, a susceptibility (10-fold) to efavirenz compared to a wildtype control RTV. On a wild type background (i.e. L100I mutation alone)displayed a moderate loss of susceptibility (10-fold) and a slight lossof susceptibility (3-fold) to nevirapine compared to a wild type controlRTV. On a wild type background (i.e. K103M mutation alone) the K103N-RTVdisplayed moderate loss of to delavirdine susceptibility (50-fold),nevirapine (55-fold) and efavirenz (30-fold) compared to a wild typecontrol RTV. The K103N-Y188L-RTV displayed substantial loss ofsusceptibility to delavirdine (greater than 250-fold), nevirapine(greater than 800-fold) and efavirenz (greater that 250-fold) comparedto a wild control RTV. The L100I-K100I-RTV displayed substantial loss ofsusceptibility (greater that 250-fold) to delavirdine and efavirenz(greater that 250-fold) and a moderate loss of susceptibility (70-fold)to nevirapine compared to a wild type control RTV. TheL100I-K103N-Y188L-RTV displayed substantial loss of susceptibility todelavirdine (greater than 250-fold), nevirapine (greater than 250-fold)compared to a wild type control RTV. Novel combinations resulted inunpredicted resistance patterns that were different from those patternsobserved for each mutation alone.

1. A method of assessing the effectiveness of a non-nucleoside reverse transcriptase inhibitor (NNRTI) on a human immunodeficiency virus type 1 (HIV-1)-infected patient, comprising detecting in a plasma sample collected from the HIV-1-infected patient the presence of an HIV-1 reverse transcriptase (RT) nucleic acid that encodes one of the following combination of mutations: G190A/S and K101E, or G190A/S and A98G, wherein said combination of mutations is associated with increased susceptibility to delavirdine and decreased susceptibility to nevirapine and efavirenz.
 2. The method of claim 1, wherein the mutation at codon 190 encodes alanine (A).
 3. The method of claim 1, further comprising evaluating whether the nucleic acid encoding reverse transcriptase has an additional mutation K103N, wherein the presence of the additional mutation in combination with the mutations G190A/S and K101E, or G190A/S and A98G, is correlated with decreased susceptibility to delavirdine, nevirapine, and efavirenz.
 4. The method of claim 1, wherein the mutation at codon 190 encodes serine (S). 